Unleashing the Potential of Immuno-Oncology Therapies

Unleashing the Potential of Immuno-Oncology Therapies May 31, 2025 2025 Xilio Therapeutics, Inc.

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or the development of Xilio s current or future product candidates; Xilio s ability to obtain and maintain sufficient preclinical and clinical supply of current or future product candidates; Xilio s advancement of multiple early-stage

masked T cell engager programs; initial, preliminary or interim preclinical or clinical data or results, which may not be replicated in or predictive of future preclinical or clinical data or results; Xilio s ability to successfully demonstrate

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quarter of 2026, including to advance its pipeline of tumor-activated I-O molecules; the impact of international trade policies on Xilio s business, including U.S. and China trade policies; and

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other companies, which are the property of their respective owners. TECENTRIQ is a registered trademark of Genentech USA Inc., a member of the Roche Group. 2

Immuno-Oncology Therapy is the Key to Curative Potential, But Continues to Be Limited by Systemic Toxicity

Xilio believes the next revolution in I-O therapy will harness the power of the body s immune system by leveraging the dysregulated biology of the tumor against itself 3

I-O: immuno-oncology

Xilio Exploits Dysregulated MMP Activity, a Hallmark of Invasive Cancer Common Across a Wide Range of Solid

Tumors, to Activate Molecules in the Tumor MMPs are dysregulated broadly across solid tumors MMP mRNA expression in tumor vs. normal tissue MMPs MMPs and immune cells co-localize at the invasive edge of tumors

In situ mRNA expression in human breast cancer Tumor cells MMP T cells (TROP2) (MMP2) (CD4, CD8A) intensity tracks with log2-transformed fold changes (log2FC). Pre-processed TCGA data were obtained from UCSC

Xena. Right panel: Spatial gene expression analysis using Xenium platform (10X Genomics) showing expression of TROP2 (TACSTD2, pink), MMP2 (yellow), CD4 and CD8A (blue) in a human breast cancer sample. https://www.10xgenomics.com/products/xenium-in- 4 situ/human MMP: matrix -breast metalloproteases -dataset-explorer; Xenium Explorer Version 1.2.0; Instrument Analysis Version: Xenium- 1.0.1

Xilio Platform and Capabilities Delivering a Portfolio of Differentiated Molecules Across Diverse Mechanisms

and Architectures T Cell Antibodies Cytokines Bispecifics Engagers Vilastobart XTX301 XTX501 Tumor-Activated Cell Engagers Tumor-activated Tumor-activated IL-12 Tumor-activated

PD-1/IL-2 bispecific PSMA, CLDN18.2, STEAP1 Fc-enhanced anti-CTLA-4 and AbbVie Program

Clinically Validated Platform Technology and Capabilities Proprietary masking libraries and custom computational design workflows Clinically validated protease cleavage elements tumor-selective activation of molecules (250+ patients

enrolled to date across clinical programs) Proprietary preclinical and clinical translational models Highly developable architectures with low immunogenicity in clinic and excellent stability

Top-Tier Pharma Partnerships and Collaborations Significant future contingent payments, Co-funded clinical supply including:* $17.5M development milestone prior to opt-in (Gilead) Near-term collaboration milestones (AbbVie) Option to license IL-12 Nomination fees for additional programs (AbbVie) $75M option fee on Phase 1/2 data package

(Gilead) Multi-program collaboration and option to license tumor-activated cell engagers *Cash runway currently anticipated into Q1 2026, prior to and any potential future contingent payments under AbbVie and Gilead agreements. 5 PSMA:

prostate-specific membrane antigen; CLDN18.2: claudin 18.2; STEAP1: six-transmembrane epithelial antigen of prostate 1

Advancing Pipeline of Tumor-Activated Immunotherapies Leveraging Clinically-Validated Platform Technology,

Including Masked T Cell Engagers Collaborations and Program Tumor Types Discovery IND-Enabling Phase 1 Phase 2 Phase 3 Partnerships Vilastobart (1) Metastatic Tumor-activated anti-CTLA-4 MSS CRC Co-funded clinical supply XTX301 (2) Advanced Tumor-activated IL-12 Solid Tumors Option to license XTX501 Advanced

Tumor-activated PD-1/IL-2 Solid Tumors PSMA Prostate Masked T cell engager CLDN18.2 Gastric, Pancreatic, Masked T cell engager Esophageal, Lung STEAP1 Prostate, Lung

Masked T cell engager Colorectal Undisclosed Undisclosed Masked T cell engagers (3) Collaboration and option 1 2. . Evaluating Evaluating vilastobart XTX301 in in Phase combination 1 monotherapy with atezolizumab dose escalation

(Tecentriq ) and dose in expansion patients with for metastatic the treatment MSS of CRC advanced under solid co-funded tumors clinical under trial exclusive supply global agreement partnership with Roche

with Gilead . . 3. Advancing an initial masked T cell engager program in collaboration with AbbVie, and AbbVie has the right to nominate up to two additional masked T cell engager programs. 6 CRC: colorectal cancer; MSS: microsatellite stable

Anticipated Milestones* Anticipated Milestone Timing Vilastobart (tumor-activated anti-CTLA-4) Reported updated Phase 2 data in combination with atezolizumab in MSS CRC May 2025 (ASCO) Report updated Phase 2 data in combination with atezolizumab in MSS CRC, including at the 150 mg Q6W dose level

for vilastobart 1H 2026 XTX301 (tumor-activated IL-12) Development milestone ($17.5M) prior to potential Phase 1/2 opt-in Undisclosed XTX501 (tumor-activated PD-1/IL-2) IND submission Mid 2026 Masked T cell engager programs Nominate development candidate for PSMA Q3 2025 Nominate development candidate for CLDN18.2 Q4 2025 Nominate

development candidate for STEAP1 1H 2026 IND submissions for at least two masked T cell engager programs 2027 *Cash runway currently anticipated into Q1 2026, prior to any potential future contingent payments under AbbVie and Gilead agreements 7

Q6W: once every six weeks

Vilastobart Tumor-Activated, Fc-enhanced

Vilastobart: Tumor-Activated, High Affinity Binding, Fc-Enhanced Anti-CTLA-4 Highlights from Previously Reported Data Inactive State High affinity binding, 10x potency of ipilimumab in preclinical studies(1) Fc mutations for enhanced effector function (ADCC),

improved T cell priming and Treg Variable Domain depletion ADCC-enhanced Fc On-treatment biopsies in Phase 1 monotherapy Cleavage Sites demonstrated >70% activated molecule in tumor with <15%

activated molecule in periphery Generally well-tolerated in Phase 1/2 (both as a Masking Domain monotherapy and combination therapy) consistent with tumor-activated design Confirmed PRs observed with monotherapy and combination 1.

Ipilimumab analog used for preclinical studies 9 ADCC: antibody-dependent cell-mediated cytotoxicity; NSCLC, non-small lung cancer; PR: partial response; Treg: regulatory T cells

CRC Incidence is Increasing, Particularly In Young Adults, with New Cases Typically Identified at Later Stages

~90,000 new cases of Stage 4 CRC patients ~95% of Stage 4 CRC patients have MSS CRC (1) estimated in the US per year CRC is 2nd in cancer-related deaths in the US and leading cause of cancer-related death in

MSI-H men younger than 50 in the US (2) ~5% CRC is 3rd in total annual new cases globally, with ~1.9M new cases and ~900,000 deaths MSS CRC related to CRC globally (3) ~95% 1. Kawazoe. J Clin Oncol.

2024;42:2918. 2. Siegel. CA Cancer J Clin.2023;73:233. 3. Bray. CA Cancer J Clin 2024;74:229. 10 MSI-H: microsatellite instability-high

Approved I-O Therapies Have Shown Little to No Efficacy in MSS CRC,

While Other Anti-CTLA-4 Molecules in Development Have Been Limited by Toxicity to Date Treatment for advanced MSS CRC typically includes Current Standard of Care in 3L+ MSS CRC Provides Minimal Benefit

chemotherapy combinations, (1) followed by clinical trials or late-line therapies with minimal benefit (OS: ~6-9 Treatment Line ORR% months) (2) Regorafenib 3L/4L+ ~1% Immune checkpoint inhibitors

(pembrolizumab/ Fruquintinib 3L/4L+ ~1.5% nivolumab) approved in MSI-H CRC have no meaningful Lonsurf 3L/4L+ ~1.5% efficacy in patients with MSS CRC (0-3% ORR) (3)t

Lonsurf + Avastin 3L ~6% Atezolizumab (PD-1) demonstrated no meaningful monotherapy efficacy in patients with MSS CRC ( 2% ORR) Third party anti-CTLA-4 agents in development: Fc-enhanced, not masked anti-CTLA-4 reported 8-19% ORR in patients with MSS CRC without liver metastases but up to

~30% discontinuation rates due to AEs (4) Not Fc-enhanced, masked anti-CTLA-4 reported 17-24% ORR in patients with MSS CRC

without liver metastases but up to ~40% Grade 3+ treatment-related AEs (5) 2024;42:2918 1. Eng. Lancet . .3 2024;404:294 . Sahin. Am Soc . 2. Clin Grothey Oncol . Lancet Educ .Book 2013;381:303; . 2022:42:1 Mayer . 4. Phase . N Engl 2 response J

Med. 2015;372:1909; data and Phase Li 1. safety JAMA .data 2018;319:2486; reported by Dasari Agenus . Lancet Inc. for. 2023;402:41; the combination Kawazoe of botensilimab . J Clin Oncol (anti.- 11 CTLA 3L: third

-4) line; and balstilimab 4L: fourth line; (PD AE: -1). 5 adverse . Phase event; 2 data ORR: by Adagene objective Inc response . for the combination rate; OS: overall of

muzastotug survival (anti-CTLA-4) and pembrolizumab (PD-1).

Vilastobart Has Demonstrated Meaningful Clinical Activity and Differentiated Safety Profile in Combination,

Supporting Opportunity in MSS CRC and a Range of Other Tumor Types 26% preliminary ORR observed in Phase 2 in heavily pre-treated patients (80% of patients 3L+) with MSS CRC without liver metastases Deep and

durable responses ongoing for up to 37 weeks as of the data cutoff Combination of vilastobart and atezolizumab continued to demonstrate differentiated safety and tolerability profile Low incidence of colitis and other immune-mediated AEs, which have

limited the potential for other anti-CTLA-4 agents Significant opportunity for vilastobart as a combination treatment in multiple tumor types 12 Data cutoff date: May 12, 2025

Vilastobart (anti-CTLA-4) Phase 2 Data for Combination of Vilastobart

and Atezolizumab Presented at ASCO on May 31, 2025

Phase 1/2 Study Design for Vilastobart in Advanced Solid Tumors and Metastatic MSS CRC KEY PHASE 2 ELIGIBILITY

PHASE 1A/B PHASE 1C PHASE 2 At least 1 prior chemotherapy Monotherapy Dose-Escalation Vilastobart + Atezolizumab Vilastobart + Atezolizumab regimen for metastatic CRC and Expansion Dose-Escalation No prior checkpoint inhibitors Advanced Solid Tumors

Advanced Solid Tumors Metastatic MSS CRC (n=39) (n=26) (n=44) Must be assessed for MSI-H or dMMR status patients Vilastobart monotherapy RP2D Vilastobart dose levels up to Vilastobart 100 mg Q6W + with MSI-H/dMMR tumors established as 150 mg Q6W 150 mg Q6W recently cleared atezolizumab 1200 mg Q3W are excluded Enrollment completed Evaluation of patients ongoing Enrolling ~10 patients at vilastobart 150 mg Q6W to

evaluate efficacy at higher dose level 14 NCT04896697 Q3W: once every three weeks

Phase 2 Enrolled Heavily Pre-Treated (3L+) Patients With MSS CRC With

and Without Liver Metastases, Including Patients With Peritoneal Metastases Patient Total Prior Lines of Anti-Cancer Tumor Total Treatment Total Characteristics (n=44) Treatment Types (n=44) Status (n=44) Age, median (range) 55 (25-82) Median 4 (1-8) MSS CRC 44 Continuing on Treatment 14 Female 22 (50%) 1 4 (9%) with liver metastases 17 Discontinued Treatment 30 ECOG PS 0 18 (41%) 2 5 (11%) without

liver Disease Progression 23 27 ECOG PS 1 26 (59%) metastases Adverse Events 4 with peritoneal Investigator Decision 1 8 metastases Other 2 80% of patients had 3 or more prior lines of treatment 15 Data cutoff date: May 12, 2025

Preliminary 26% ORR Reported in Patients With MSS CRC Without Liver Metastases 200% 80 Best Response

Without With Best Response Liver Metastasis Liver Metastasis 60 Partial Response Per RECIST (n = 27) (n = 17) PR 7* 0 in 41% 40% Stable Disease 40 SD 5 3 Disease Progression ORR 26% 0% lesions 30% 25% 22% baseline 20 16% +20% 10% 10% 9% target 8% 7%

3% 2% 0% 0% fromof 0 -2% change -20 diameters -30% % -40

-32% of # -42% Best -47% -52% sum -60 -54% # -61% -71% -80 * Responders include: patients with both KRAS mutant and KRAS wild type

tumors a patient with peritoneal metastases Data # PR cutoff confirmed date: after May the 12, data 2025 cutoff date * 6 confirmed PRs, 1 unconfirmed PR. In figure above, unconfirmed PR is noted with an asterisk. Figure above represents best

response in target lesions. 3 patients with MSS CRC without liver metastases not included in figure above had progressive disease (patients had new 16 lesions but target lesions were not assessed).

Durable Anti-Tumor Activity Observed in Patients With MSS CRC Without Liver Metastases 6 out of 7 responders

remain on treatment Treatment duration currently up to 37 weeks Best Response Partial response Stable disease Disease progression New lesion Treatment Status Ongoing Discontinued # Data cutoff date: May 12, 2025 17 # PR confirmed after the data

Significant Reductions in Tumor Burden With Meaningful Improvements in Clinical Symptoms and Decreases in Tumor

Biomarkers Data cutoff date: May 12, 2025 18 CEA: carcinoembryonic antigen; ctDNA: circulating tumor DNA

Radiographic Responses Were Accompanied by Substantial ctDNA Reductions Changes in sum of diameters Changes in

ctDNA score 200% Baseline Best Baseline % from in Diameters of ctDNA Change Change Sum % in from Best Score * Data cutoff date: May 12, 2025 Dodged waterfall plot showing best % change from baseline in the sum of diameters of target lesions

(left y-axis; purple bars) and best % change from baseline in ctDNA score (right y-axis; 19 yellow bars). Each pair of bars corresponds to the same patient (x-axis).

MSS CRC Patient With Liver Metastasis and Previously Reported Confirmed PR From Phase 1C Still On Treatment

After 14+ Months With Response Deepening Patient with MSS CRC and Liver Metastasis Screening 9 weeks 18 weeks 27 weeks 36 weeks 45 weeks 57 weeks Sum of diameters 98.4 mm 70.5 mm 71.0 mm 66.3 mm 68.9 mm 59.2 mm 58.8 mm

Change 28% 28% 33% -30% -40% -40% 69 year-old female Target Liver Lesion

Baseline Target Liver Lesion 9 Weeks 5 prior lines of therapy: FOLFOX-Avastin 15.6 mm 6.8 mm FOLFIRI-Avastin Cetuximab Lonsurf Target Liver Lesion 18 Weeks Target Liver Lesion 27

Weeks FOLFIRI-Panitumamab Phase 1C (combination dose escalation) 5.8 mm No visible lesion Administered vilastobart (150 mg Q6W) + atezolizumab (1200 mg Q3W) 20