ATG101 Antibody

What is the structural design of ATG-101?

ATG-101 is engineered as a tetravalent "2+2" PD-L1×4-1BB bispecific antibody. It was developed by introducing lower affinity 4-1BB scFv into a human IgG1 PD-L1 monoclonal antibody . The design includes specific mutations on the CH2 domain that abolish binding capacity to most FcγRs while maintaining binding to FcγRn, which is critical for its pharmacokinetic properties . This engineering approach creates an antibody with dual targeting capabilities while minimizing off-target effects.

How does ATG-101 bind to its target molecules?

ATG-101 binds PD-L1 and 4-1BB concurrently, with a greater affinity for PD-L1 . This asymmetric binding profile is deliberately designed to ensure that the antibody first localizes to PD-L1-rich tumor microenvironments before engaging with 4-1BB on immune cells . The higher affinity for PD-L1 ensures preferential binding to tumor cells expressing this marker, which is fundamental to its tumor-focused mechanism of action.

How does ATG-101 activate T cells differently from conventional 4-1BB agonists?

ATG-101 activates 4-1BB+ T cells specifically when cross-linked with PD-L1-positive cells, unlike conventional 4-1BB agonists that activate T cells indiscriminately . In in vitro studies, ATG-101 activated CD8+ T lymphocytes to produce IL2 at concentrations of 0.1 and 1 nmol/L, with cytokine production positively correlating with PD-L1 expression levels . Importantly, when HEK293 cells expressing PD-L1 were cocultured with parental HEK293 cells in various proportions, ATG-101-induced IFNγ release increased with the proportion of PD-L1+ cells, while no activation occurred in the absence of PD-L1 cross-linking . This PD-L1-dependent activation mechanism explains both its efficacy and improved safety profile.

What cellular pathways does ATG-101 influence in the tumor microenvironment?

ATG-101 triggers multiple cellular pathways in the tumor microenvironment. Gene ontology enrichment analysis revealed that interferon response, IL1 synthesis, and antigen processing and presentation signaling pathways are activated following ATG-101 treatment . Within macrophage populations, ATG-101 increases expression of M1 macrophage-associated genes (Fcgr1, Cd86, Stat1) while decreasing M2 macrophage-associated genes (Csf1r, Mrc1), shifting the balance toward pro-inflammatory, anti-tumor responses . Additionally, ATG-101 treatment leads to increased expression of cytotoxicity-related genes like granzyme B in various T-cell subsets, coupled with decreased expression of T-cell exhaustion markers such as Tox .

What tumor models are most appropriate for studying ATG-101 efficacy?

Several tumor models have demonstrated ATG-101's efficacy in preclinical research:

• MC38-hPD-L1 tumors in h4-1BB knock-in mice - useful for basic efficacy and pharmacodynamic studies

• EL-4 lymphoma model - showed significant tumor growth inhibition at doses of 3.25 and 13 mg/kg

• MC38 tumor model progressing on anti-PD-L1 treatment - critical for studying acquired resistance to checkpoint inhibitors

• Various syngeneic tumor models resistant to PD-1/PD-L1 inhibitors - valuable for studying innate resistance mechanisms

Researchers should select models based on their specific research questions, particularly considering whether they are investigating innate or acquired resistance to immunotherapy.

What biomarkers should be monitored when evaluating ATG-101's activity?

Based on published research, the following biomarkers should be monitored when evaluating ATG-101:

• T-cell populations:

  • CD8+ T cell infiltration and proliferation

  • CD8+ T/Treg ratio in tumor vs. peripheral tissues

  • Effector memory T cell levels

  • Late activated T cells and NK T cells

• Molecular markers:

  • Granzyme B (Gzmb) expression in T-cell subsets

  • T-cell exhaustion markers (e.g., Tox)

  • Immunosuppressive chemokines (Xcl1, Ccl1)

  • 4-1BB expression across T-cell populations

  • TRAF expression (downstream of 4-1BB signaling)

• Cytokine profiles:

  • IL2 production

  • IFNγ levels and related gene expression

How effective is ATG-101 in tumors that have progressed on checkpoint inhibitor therapy?

ATG-101 demonstrates significant efficacy in tumors that have progressed on checkpoint inhibitor therapy. In a MC38 tumor model that progressed on anti-PD-L1 treatment (atezolizumab), switching to ATG-101 induced growth inhibition or regression in these resistant tumors . Mice switched from atezolizumab to ATG-101 showed a significant survival advantage over those remaining on atezolizumab or receiving placebo . Multiplex immunofluorescence staining revealed that ATG-101 significantly augmented CD8+ T cells in the tumor microenvironment compared to continuing atezolizumab treatment . This indicates ATG-101's potential utility in patients who have developed acquired resistance to standard checkpoint inhibitor therapy.

What mechanisms enable ATG-101 to convert "cold" tumors to "hot" tumors?

ATG-101 transforms immunologically "cold" tumors to "hot" tumors through several key mechanisms:

• Enhanced T cell activity:

  • Increased proliferation of CD8+ T cells

  • Greater infiltration of effector memory T cells

  • Improved CD8+ T/Treg ratio in the tumor microenvironment

• Altered immune cell interactions:

  • Comprehensive single-cell RNA sequencing revealed that ATG-101 treatment creates 21 unique T-cell-dendritic cell interactions not present in control samples

  • These include four ICAM1-involved interactions crucial for CD8+ T-cell memory and activation

  • Elimination of tumor-promoting interactions such as CCR5/CCL8, FN1/integrin, and TREM2/APP

• Creating positive feedback loops:

  • Increased PD-L1 expression on dendritic cells after treatment

  • Elevated 4-1BB expression on T cells

  • These changes amplify T cell-dendritic cell cross-linking over time, further activating 4-1BB signaling

How does ATG-101 mitigate the hepatotoxicity concerns associated with conventional 4-1BB agonists?

ATG-101 addresses hepatotoxicity concerns through its unique PD-L1-dependent activation mechanism:

• Localized activation: By requiring PD-L1 cross-linking for 4-1BB activation, ATG-101 localizes its activity primarily to PD-L1-rich tumor tissues rather than healthy tissues .

• Anti-inflammatory hepatic environment: Studies revealed that ATG-101 treatment leads to increased Treg levels and decreased CD8+ T/Treg ratios in blood and liver, suggesting an anti-inflammatory state that reduces the risk of liver damage .

• Empirical evidence: ATG-101 was well tolerated without inducing hepatotoxicity in non-human primates in Good Laboratory Practice (GLP) toxicology studies .

This improved safety profile is critical, as hepatotoxicity has been a major obstacle in the clinical development of previous 4-1BB agonists .

What dosing considerations are important for ATG-101 to maintain efficacy while minimizing toxicity?

Optimal dosing of ATG-101 should consider:

• Computational modeling findings: Semimechanistic pharmacology modeling indicates that both 4-1BB/ATG-101/PD-L1 trimer formation and PD-L1 receptor occupancy are maximized at approximately 2 mg/kg of ATG-101 .

• PD-L1 expression levels: In vitro studies showed that ATG-101-induced cytokine production correlates with PD-L1 expression levels. While minimal proinflammatory cytokines were produced when co-cultured with cells expressing moderate PD-L1 levels, significantly higher levels occurred with cells expressing much more PD-L1 .

• In vivo efficacy data: In the EL-4 lymphoma model, ATG-101 at 3.25 and 13 mg/kg (administered once every 3 days) exhibited significant tumor growth inhibition of 35% and 50%, respectively .

How might ATG-101 be studied in combination with other immunotherapies?

ATG-101's unique mechanism suggests several promising combination approaches:

• With other checkpoint inhibitors: Since ATG-101 already contains anti-PD-L1 activity, combinations with inhibitors targeting different checkpoints (CTLA-4, LAG-3, TIM-3) could provide complementary immune activation pathways.

• With T-cell engagers: ATG-101's ability to activate 4-1BB signaling might synergize with bispecific T-cell engagers that bring T cells in proximity to tumor cells.

• With cancer vaccines: ATG-101's enhancement of antigen presentation pathways suggests potential synergy with cancer vaccines that provide tumor-specific antigens.

Research designs should include appropriate controls and sequential treatment arms to determine optimal timing and sequencing of combination therapies.

What single-cell analysis approaches reveal the most about ATG-101's effects on the tumor microenvironment?

Single-cell RNA sequencing offers critical insights into ATG-101's mechanisms:

• Cell-cell interaction analysis: Using tools like CellPhoneDB to analyze T-cell-dendritic cell communications revealed that ATG-101 treatment created 21 unique interactions not present in control samples, while eliminating 14 potentially tumor-promoting interactions .

• Gene expression profiling: Analyzing expression patterns of immune-related genes across different cell subpopulations revealed ATG-101's diverse effects on various immune cell types .

• Pathway enrichment analysis: Gene ontology enrichment of upregulated genes identified activation of interferon response, IL1 synthesis, and antigen processing pathways .

Researchers should consider designing comprehensive single-cell experiments that sample the tumor microenvironment at multiple timepoints to capture the dynamic changes induced by ATG-101 treatment.

What predictive biomarkers might identify patients most likely to respond to ATG-101?

Based on ATG-101's mechanism of action, potential predictive biomarkers include:

• PD-L1 expression levels: Since ATG-101 requires PD-L1 cross-linking for activation, tumors with higher PD-L1 expression may show better responses .

• 4-1BB expression on tumor-infiltrating lymphocytes: Higher baseline expression may predict stronger activation potential .

• Baseline T-cell exhaustion profile: ATG-101's ability to activate exhausted T cells suggests patients with T-cell exhaustion rather than T-cell absence might benefit most .

Translational research should correlate these biomarkers with treatment outcomes to develop clinically useful predictive tools.

What are the key pharmacodynamic endpoints for early-phase clinical trials of ATG-101?

Early-phase clinical trials should consider these pharmacodynamic endpoints:

• Immune cell monitoring:

  • Changes in tumor-infiltrating CD8+ T cells

  • Alterations in CD8+/Treg ratios in tumor biopsies versus peripheral blood

  • Changes in effector memory T-cell populations

• Molecular markers:

  • Changes in granzyme B expression in T cells

  • Alterations in exhaustion markers like Tox

  • 4-1BB receptor occupancy

• Functional assessments:

  • T-cell activation and proliferation markers

  • Cytokine profile changes

  • Evidence of PD-L1/4-1BB cross-linking