# Tislelizumab: Integrated Analysis of Tumor, Peripheral Blood, and Patient Treatment History

**Desamparados Roda , Alexander Chung , Spencer Huggett , Jon Zugazagoitia , Elena Garralda , Alexander I. Spira , Yujie Zhao , Peter J. Oppelt , Anthony B. El-Khoueiry ,**  
**Margaret E. Chen , Chunjie Jiang , Melissa Harney , Hombline Poullain , Germain Margall-Ducos , Eladio Marquez , Francisco Adrian , Jinping Gan**  
Hospital Clínico Universitario de Valencia , HiFiBiO Therapeutics Inc. , Hospital Universitario 12 de Octubre , Vall d’Hebron Institute of Oncology , NEXT Oncology Virginia , Mayo Clinic Florida , Washington University School of Medicine in St. Louis , University of Southern California Norris Comprehensive Cancer Center

## BACKGROUND

• Agonism of tumor necrosis factor receptor-2  
(TNFR2) enhances anti-tumor immunity by  
stimulating T- and NK-cells in the tumor  
microenvironment.

• Emunkitug (HFB200301), an anti-TNFR2 agonistic  
monoclonal antibody, triggers both innate and  
adaptive immune responses.

• Previously we reported Phase 1 results that  
demonstrated tolerable safety profile and clinically  
meaningful efficacy with emunkitug as  
monotherapy and in combination with tislelizumab  
(TIS) in advanced refractory solid tumors.

• Model-informed optimization of dosing led to Q2W  
regimen in addition to Q4W.

• Here, we report updated safety and efficacy results  
with longer follow-up time.

• In addition, we report potential biomarkers  
predictive of response to emunkitug combined with  
TIS.

## STUDY DESIGN and PATIENT DEMOGRAPHICS

## Study Design

DL (Dose Level); HFB (HFB200301); Q4W (once every 4 weeks); TIS (tislelizumab)

## Demographics and Clinical Characteristics

| Characteristic | Monotherapy(n=38) | Combination(n=34) |
| --- | --- | --- |
| Median age,years(range) | 61(21-77) | 62.5(18-81) |
| Sex,n(%) |  |  |
| Female | 18(47) | 12(35) |
| Male | 20(53) | 22(65) |
| ECOG PS,n(%) |  |  |
| 0 | 14(37) | 13(38) |
| 1 | 24(63) | 21(62) |
| Median time since initial diagnosis(range),years | 2.0(0.3-22.0) | 3.2(0.5-15.9) |
| Number of prior systemic cancer therapy regimens,n(%) |  |  |
| Median(range) | 2(1-4) | 2(1-4) |
| 1 | 9(24) | 4(12) |
| 2 | 13(34) | 15(44) |
| ≥3 | 16(42) | 15(44) |
| Received prior anti-PD-(L)1 therapy,n(%) |  |  |
| Yes | 25(66) | 30(88) |
| No | 13(34) | 4(12) |
| Median follow-up time,months(range) | 2.0(0.5-7.3) | 2.3(0.6-13.9+) |
| Tumor types,n(%) |  |  |
| Clear cell renal cell carcinoma | 3(8) | 8(23) |
| Cervical cancer | 2(5) | 5(15) |
| Gastric cancer,EBV+ | 0(0) | 1(3) |
| Head and neck squamous cell carcinoma | 5(13) | 3(9) |
| Melanoma | 3(8) | 5(15) |
| Non-small cell lung cancer | 7(19) | 7(20) |
| Pleural mesothelioma | 5(13) | 3(9) |
| Sarcoma | 11(29) | 1(3) |
| Testicular germ cell tumor | 2(5) | 1(3) |

EBV+, Epstein-Barr virus positive; ECOG PS, Eastern Cooperative Oncology Group performance status; PD-(L)1, programmed cell death protein (ligand) 1.

## SAFETY PROFILE

## Safety Summary of HFB200301 Q2W/Q4W ± TIS Q4W

**Emunkitug Q4W/Q2W ± TIS demonstrated tolerable safety profile**  
• **TRAEs mostly limited to Grade 1-2, including TRAEs of interest (inflammatory and cutaneous toxicity)**  
• **No grade 4 or 5 TRAE and no TRAE leading to discontinuation or dose reduction**

| Treatment-related AE (TRAE) | Emunkitug Q4W(N=27) | Emunkitug Q2W(N=11) | Emunkitug Q4W+TIS(N=12) | Emunkitug Q2W+TIS(N=22) |
| --- | --- | --- | --- | --- |
| Overall TRAE, % | 13(48) | 6(55) | 8(67) | 17(77) |
| Grade 3 | 0 | 0 | 0 | 2(9) |
| Inflammatory AE, % | 4(15) | 3(27) | 4(33) | 11(50) |
| Grade 3 | 0 | 0 | 0 | 0 |
| Cutaneous Tox, % | 4(15) | 0 | 2(17) | 6(27) |
| Grade 3 | 0 | 0 | 0 | 1(5) |
| TRAE leading to discontinuation | 0 | 0 | 0 | 0 |
| TRAE leading to death | 0 | 0 | 0 | 0 |

Cutoff: 26-Sep-2025

## PRELIMINARY ANTI-TUMOR RESPONSE

## Emunkitug + TIS Combination Efficacy

**Clinically meaningful responses demonstrated by emunkitug + TIS**  
• **Figure A: Partial responses in multiple indications – mesothelioma, non small cell**  
**lung cancer, and clear cell renal cell carcinoma**  
• **All responders had prior immunotherapy and were heavily pretreated**  
• **Disease control rate (DCR) per RECIST 1.1 of 50% in Q2W and 36% in Q4W**  
**combination**  
• **Figure B: Durable clinical benefit (partial responses and stable disease per RECIST**  
**1.1) observed**

Note: 5 subjects not shown in Fig A and B had clinical progressions and did not have  
radiographic assessment of their target lesions  
Cutoff: 29-Sep-2025

## Spatial Proximity Analysis – Tumor mIF

**Spatial proximity to tumor with TNFR2+ and PD1+ immune cells**

## Table 2: RECIST 1.1 BOR vs Preceding Therapy  
## HFB200301 Q4W/Q2W + TIS (n=22)

• **Figure A: Graphical representation of mIF-derived analysis of spatial proximity**  
**to determine distance between the target cells (e.g. TNFR2+CD8 T cells) and**  
**tumor cells**  
• **Figure B: Distribution of all target cells’ distance from tumor cells (each row**  
**represents individual subject)**  
• **Figure C and D: Representative images of subjects with close (Fig C) and**  
**distant (Fig D) median tumor cell distance to TNFR2+ CD8T**

**A**

**B**

**D** Emunkitug + TIS  
Testicular cancer  
**Progressive disease**  
Median distance: **1,328 μm**

## POTENTIAL BASELINE BIOMARKERS PREDICTIVE OF RESPONSE TO EMUNKITUG + TIS

## Tumor – mIF: Spatial Proximity of TNFR2+CD8 or TNFR2+NK Cells to Tumor Cells as Potential Response Predictive Biomarker

## and NK cells to enhance cytotoxic antitumor responses

**Closer proximity of tumor cells with TNFR2+ CD8 and TNFR2+ NK cells associated with prolonged progression-free survival (PFS) in response to emunkitug + TIS**  
• **Figure A: Emunkitug-mediated activation of TNFR2+ CD8 T cells in closer proximity to tumor cells may be predictive of response**

**mIF Spatial Proximity – Emunkitug + TIS:**  
**TNFR2+ CD8T – Tumor cells**

**A**

Cutoff: 29-Sep-2025

## Periphery – Single-cell RNA & Flow Cytometry: NK Abundance as Potential Response Predictive Biomarker

**High peripheral levels of suppressive subset of cytotoxic NK cells at baseline may predict clinical benefit to emunkitug + TIS**  
• **Figure A: scRNA analysis revealed that suppressive subset of cytotoxic NK cells may predict clinical benefit (PR or SD per RECIST 1.1) to emunkitug + TIS**  
• **Figure B: Correlation between scRNA versus flow cytometry levels of suppressive subset of NK cells**

## Monotherapy Emunkitug Q4W (n=27)

• **Figure C and Table 1: Flow cytometry analysis showed that higher peripheral levels of suppressive subset of NK cells may be predictive of prolonged progression-free survival (PFS) and**  
**improved DCR (PR+SD)**

**A**

**Cytotoxic NK cell count – All subjects:**  
**scRNA vs Flow**  
**B**

**Flow cytometry – Emunkitug + TIS:**  
**Suppressive subset of cytotoxic NK vs PFS**

**C**

## Monotherapy Emunkitug Q2W (n=11)

**Table 1: RECIST 1.1 BOR vs Suppressive**  
**Subset of Cytotoxic  NK**

| HFB200301 Q4W/Q2W+TIS(n=26) |  |  |
| --- | --- | --- |
| RECIST 1.1 | NK-Lown=18 | NK-Highn=8 |
| PR | 1 | 1 |
| SD | 4 | 4 |
| PD | 13 | 3 |
| DCR(PR+SD) | 5(28) | 5(63) |
| ORR | 1(6) | 1(13) |
| Median Prior LOT | 2.5(1-4) | 2(1-4) |

Cutoff: 29-Sep-2025

## Patient Record: Immediate Prior Immunotherapy May be a Surrogate for Tumor and Peripheral Biomarkers

**Immediate prior immunotherapy may be predictive of response to emunkitug + TIS combination**

• **Figure A and B: Subjects with immediate prior immunotherapy may be linked to closer proximity of TNFR2+CD8/NK to tumor cells and cytotoxic NK cells in the periphery at baseline**  
• **Figure C and Table 2: Immediate prior immunotherapy may be predictive of prolonged PFS and increased DCR (PR+SD) amongst patients with indications of interest**

**Preceding therapy: C,** Chemotherapy; **T,** Targeted**; IO,** Immunotherapy

## IN DEPTH LOOK AT NSCLC

Cutoff: 29-Sep-2025

**NOTE:** Included in Figure C and Table 1 are indications of interest (ccRCC, gastric, HNSCC, mesothelioma, NSCLC)

## Clinical and Biomarker Data Support Further Development in NSCLC

| RECIST 1.1 | Others(chemo,targeted)n=12 | IO(+/- chemo or targeted)n=10 |
| --- | --- | --- |
| PR | 2 | 1 |
| SD | 4 | 8 |
| PD | 6 | 1 |
| DCR(PD+SD) | 6(50) | 9(90) |
| ORR | 2(17) | 1(10) |
| Median Prior LOT | 3(1-4) | 2.5(1-4) |

• **Compared to 2L standard-of-care docetaxel +/- ramucirumab which reported**  
**median PFS of 2.7 – 4.5**

**Early signs of improvement in PFS with emunkitug + aPD-1 in NSCLC patients,**   
**especially in biomarker enriched patients**

*Median prior LOT (3, range: 2-4); all had prior anti-PD-(L)1

|  | 3L+IO-refractory NSCLC* |  |
| --- | --- | --- |
|  | Overall(n=7) | Biomarker+(n=5) |
| ORR | 14% | 20% |
| DCR | 71% | 80% |
| mPFS | 7.9mo | 10.5+mo |

**Biomarker+:** Closer TNFR2+CD8—Tumor proximity and/or higher peripheral cytotoxic NK

## RECIST 1.1 Efficacy: Emunkitug + TIS

**Biomarker-:** Neither

## CONCLUSIONS

• Longer follow-up continued to show tolerable safety profile and durability of the  
partial responses and stable diseases in response to emunkitug + TIS.

• Integrative analysis of tumor, peripheral blood, and patient records (treatment  
history / preceding therapy) identified potential biomarkers predictive of clinical  
benefit and prolonged PFS in response to emunkitug + TIS.

• Predictive biomarkers in the tumors (TNFR2+CD8T in close proximity to tumor) and  
periphery (higher levels of suppressive subset of cytotoxic NK cells) align with  
emunkitug’s MOA in activating CD8 T and NK cells

• Suggests that the combination efficacy is dependent on emunkitug-mediated  
activation of CD8 T and NK cells in addition to PD-1 blockade

• Patient enrichment for response by NK cell count via flow cytometry may be a  
practical patient selection strategy for future development

• Early signs of clinically meaningful efficacy in NSCLC warrants further evaluation with  
additional patients

## Acknowledgments and references

| 1. | Roda, D. et al.,(2024)ASCO 2024.Chicago,IL,USA |
| --- | --- |
| 2. | Roda,D.et al.(2024)ESMO 2024.Barcelona,Spain. |
| 3. | Study sponsored by HiFIBiO Inc.