Targeting the PD-1: Biological Roles and Therapeutic Strategies

• Gene Information: The PDCD1 gene encodes the PD-1 receptor to critically regulate T-cell tolerance and prevent autoimmunity.
• Protein Expression: PD-1 is induced on activated immune cells (like T cells), while its ligands, PD-L1 and PD-L2, are often hijacked and overexpressed by tumor cells.
• Signaling Pathway: Engagement with its ligands triggers intracellular SHP-2 phosphatase recruitment, shutting down TCR/CD28 signaling (PI3K/Akt) to arrest T-cell proliferation and cytokine release.
• Therapeutic Inhibition: Therapeutic anti-PD-1/PD-L1 antibodies block this checkpoint brake, successfully reactivating tumor-reactive T cells to destroy cancer cells.

Targeting the IL2/IL15: Biological Roles and Therapeutic Strategies

• Gene Information: The IL2 and IL15 genes encode structurally related four-α-helix bundle cytokines that orchestrate lymphocyte homeostasis and immune responses.
• Protein Expression:  While IL-2 is a secreted cytokine produced mainly by activated T cells, IL-15 is predominantly membrane-bound via trans-presentation by dendritic cells, with both sharing the common βγ receptor subunits but utilizing distinct private αchains (CD25 vs CD215).
• Signaling Pathway: Both cytokines signal through the shared IL-2/15Rβγ complex to activate the downstream JAK1/JAK3-STAT5, PI3K/Akt, and MAPK pathways, driving the proliferation and survival of T cells and NK cells.
• Therapeutic Inhibition: Targeted blockade of IL-2/IL-15 signaling pathways (via antibodies against the shared β subunit or specific α chains) serves as a potent therapeutic strategy to alleviate chronic inflammation, autoimmune diseases, and T-cell-driven malignancies.

PD-1

• Human PD-1 gene encoding the extracellular region and mouse PD-1 gene encoding the transmembrane and cytoplasmic region were inserted after the initiation codon ATG of mouse PD-1 gene in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.

IL2

• The exons 1-4 of mouse Il2 gene that encode the whole molecule (ATG to STOP codon) were replaced by human counterparts in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. The promoter, 5’UTR and 3’UTR region of the mouse gene are retained. The human IL2 expression is driven by endogenous mouse Il2 promoter, while mouse Il2 gene transcription and translation will be disrupted.

IL2RA

• The exons 2-6 of mouse Il2ra gene that encode extracellular domain were replaced by human counterparts in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. The genomic region of mouse Il2ra gene that encodes cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric IL2RA expression is driven by endogenous mouse Il2ra promoter, while mouse Il2ra gene transcription and translation will be disrupted.

IL2RB

• The exons 2-8 of mouse Il2rb gene that encode extracellular domain were replaced by human counterparts in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. The genomic region of mouse Il2rb gene that encodes cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric IL2RB expression is driven by endogenous mouse Il2rb promoter, while mouse Il2rb gene transcription and translation will be disrupted.

IL2RG

• The exons 1-7 of mouse Il2rg gene that encode signal peptide, extracellular domain, transmembrane domain, cytoplasmic region  were replaced by human counterparts in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. The promoter and 5’UTR region of the mouse gene are retained. The human IL2RG expression is driven by endogenous mouse Il2rg promoter, while mouse Il2rg gene transcription and translation will be disrupted.

IL15

• The exons 3-8 of mouse Il15 gene that encode the whole molecule (ATG to STOP codon) were replaced by human counterparts in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. The promoter, 5’UTR and 3’UTR region of the mouse gene are retained. The human IL15 expression is driven by endogenous mouse Il15 promoter, while mouse Il15 gene transcription and translation will be disrupted.

IL15RA

• The exons 2-6 of mouse Il15ra gene that encode extracellular domain were replaced by human counterparts in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. The genomic region of mouse Il15ra gene that encodes cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric IL15RA expression is driven by endogenous mouse Il15ra promoter, while mouse Il15ra gene transcription and translation will be disrupted.

• The mouse IL2 was detectable in the wild-type (WT) C57BL/6JNifdc mice but not in the homozygous (HO) B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. The human IL2 was exclusively detectable in homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.

Strain specific IL2 expression analysis in homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice by ELISA. Serum were collected from wild-type mice C57BL/6 mice (+/+) (female, 6-week old, n = 3) and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 6-week old, n = 3) stimulated with anti-mCD3e antibody (7.5 μg/mice) and anti-CD28 antibody (4 μg/mice) in vivo for 2 h, and analyzed by ELISA with species-specific anti-mouse IL-2 ELISA kit (Biolegend, 431004), and anti-human IL-2 ELISA kit (Biolegend, 431804).

• Mouse PD-1 was detectable in wild-type (WT) C57BL/6JNifdc mice.

Strain specific PD-1 expression analysis in wild-type C57BL/6JNifdc mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (male, 7-week-old, n=3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse PD-1 antibody (Biolegend, 109104) and anti-human PD-1 antibody (Biolegend, 329908).

• Human PD-1 was exclusively detectable in homozygous (HO) B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.

Strain specific PD-1 expression analysis in homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice by flow cytometry. Splenocytes were collected from homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (male, 7-week-old, n = 3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse PD-1 antibody (Biolegend, 109104) and anti-human PD-1 antibody (Biolegend, 329908).

• Mouse IL2RA was only detectable in wild-type (WT) C57BL/6JNifdc mice.

Strain specific IL2RA expression analysis in wild-type C57BL/6JNifdc mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (male, 7-week-old, n = 3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse IL2RA antibody (Biolegend, 102008) and anti-human IL2RA antibody (Biolegend, 302610).

• Human IL2RA was exclusively detectable in homozygous (HO) B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.

Strain specific IL2RA expression analysis in homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice by flow cytometry. Splenocytes were collected from homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (male, 7-week-old, n = 3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse IL2RA antibody (Biolegend, 102008) and anti-human IL2RA antibody (Biolegend, 302610).

• Mouse IL2RB was only detectable in wild-type (WT) C57BL/6JNifdc mice.

Strain specific IL2RB expression analysis in wild-type C57BL/6JNifdc mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (male, 7-week-old, n = 3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse IL2RB antibody (Biolegend, 105912) and anti-human IL2RB antibody (Biolegend, 339005).

• Human IL2RB was exclusively detectable in homozygous (HO) B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.

Strain specific IL2RB expression analysis in homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice by flow cytometry. Splenocytes were collected from homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (male, 7-week-old, n = 3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse IL2RB antibody (Biolegend, 105912) and anti-human IL2RB antibody (Biolegend, 339005).

• Mouse IL2RG was only detectable in wild-type (WT) C57BL/6JNifdc mice.

Strain specific IL2RG expression analysis in wild-type C57BL/6JNifdc mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (male, 7-week-old, n = 3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse IL2RG antibody (Biolegend, 132307) and anti-human IL2RG antibody (Biolegend, 338605).

• Human IL2RG was exclusively detectable in homozygous (HO) B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.

Strain specific IL2RG expression analysis in homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice by flow cytometry. Splenocytes were collected from homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (male, 7-week-old, n=3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse IL2RG antibody (Biolegend, 132307) and anti-human IL2RG antibody (Biolegend, 338605).

• The human IL15 was exclusively detectable in homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.

Strain specific IL15 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice by ELISA. Serum was collected from wild-type C57BL/6JNifdc mice and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (Female, 6-week-old, n = 3/group,) stimulated with 350 mg/kg APAP in vivo for 18 h. Expression level of human IL15 was analyzed by ELISA (anti-human IL15 ELISA kit: R&D D1500). Values are expressed as mean ± SEM.

• The mIL15RA was detectable in wild-type (WT) mice but not in the homozygous (HO) B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. The hIL15RA was exclusively detectable in homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.

Strain specific IL15RA expression analysis in homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice by flow cytometry. Bone marrow cells were collected from wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice, and cultured in 6-well plates stimulated with GM-CSF and IL-4 for 6 days and LPS (1 μg/mL) for 18 h to induce BMDCs. Then BMDCs were collected and analyzed by flow cytometry with anti-mouse IL15RA antibody (BD, 568235) and anti-human IL15RA antibody (Biolegend, 330207).

• Frequencies of NK cells, dendritic cells, monocytes, macrophages, and neutrophils in B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice were similar to those in C57BL/6JNifdc mice. For the spleen and blood, the frequency of T cells in B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice was lower than that in C57BL/6JNifdc mice, while the frequency of B cells in B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice were higher than that in C57BL/6JNifdc mice.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, blood, and lymph nodes were isolated from C57BL/6JNifdc and B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 8-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• The frequencies of CD4+ T, CD8+ T, and Tregs in homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice were comparable to those in C57BL/6JNifdc mice, demonstrating that introduction of human PD-1, IL2RA/B/G, IL15, and IL15RA in place of their mouse counterparts does not change the overall development, differentiation or distribution of these T cell subtypes in spleen, blood, or lymph nodes.

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood. Splenocytes, blood, and lymph nodes were isolated from C57BL/6JNifdc and B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 8-week-old, n = 3). Single live cells were gated on the CD3⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• T cell activation in B-hPD-1 plus/hIL2/hIL2RA/ hIL2RB/hIL2RG/hIL15/hIL15RA mice was significantly up-regulated by anti-mCD3ε antibody and anti-mCD28 antibody.

In vitro T cell activation by anti-mCD3ε antibody with or without anti-mCD28 antibody in wild-type C57BL/6JNifdc mice and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (24h). T cells were isolated from splenocytes of C57BL/6JNifdc and B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 13-week-old, n=3), and incubated in the presence of anti-mCD3ε antibody (2 ug/ml, BioXcell, BE0001-2), with or witnout anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 24h.

• T cell activation in B-hPD-1 plus/hIL2/hIL2RA/ hIL2RB/hIL2RG/hIL15/hIL15RA mice was significantly up-regulated by anti-mCD3ε antibody and anti-mCD28 antibody.

In vitro T cell activation by anti-mCD3ε antibody with or without anti-mCD28 antibody in wild-type C57BL/6JNifdc mice and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (48h). T cells were isolated from splenocytes of C57BL/6JNifdc and B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 13-week-old, n=3), and incubated in the presence of anti-mCD3ε antibody (2 ug/ml, BioXcell, BE0001-2), with or witnout anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 48h.

• T cell activation in B-hPD-1 plus/hIL2/hIL2RA/ hIL2RB/hIL2RG/hIL15/hIL15RA mice was significantly up-regulated by anti-mCD3ε antibody and anti-mCD28 antibody.

In vitro T cell activation by anti-mCD3ε antibody with or without anti-mCD28 antibody in wild-type C57BL/6JNifdc mice and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (72h). T cells were isolated from splenocytes of C57BL/6JNifdc and B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 13-week-old, n=3), and incubated in the presence of anti-mCD3ε antibody (2 ug/ml, BioXcell, BE0001-2), with or witnout anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 72h.

• T cell proliferation was tested by flow cytometry.

Quantification of T cell proliferation in vitro by anti-CD3ε antibody with or without anti-mCD28 antibody in wild-type C57BL/6JNifdc mice and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (24h). T cells were isolated from splenocytes of C57BL/6JNifdc and B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 13-week-old, n=3), and incubated in the presence of anti-mCD3ε antibody (2 ug/ml, BioXcell, BE0001-2), with or witnout anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 24h.

• T cell proliferation was tested by flow cytometry.

Quantification of T cell proliferation in vitro by anti-CD3ε antibody with or without anti-mCD28 antibody in wild-type C57BL/6JNifdc mice and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (48h). T cells were isolated from splenocytes of C57BL/6JNifdc and B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 13-week-old, n=3), and incubated in the presence of anti-mCD3ε antibody (2 ug/ml, BioXcell, BE0001-2), with or witnout anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 48h.

• T cell proliferation was tested by flow cytometry.

Quantification of T cell proliferation in vitro by anti-CD3ε antibody with or without anti-mCD28 antibody in wild-type C57BL/6JNifdc mice and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (72h). T cells were isolated from splenocytes of C57BL/6JNifdc and B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 13-week-old, n=3), and incubated in the presence of anti-mCD3ε antibody (2 ug/ml, BioXcell, BE0001-2), with or witnout anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 72h.

• Under in vitro anti-CD3 and anti-CD28 antibody co-stimulation, wild-type C57BL/6JNifdc and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice showed comparable secretion levels of IL-2 and IFN-γ.

In vitro cytokine production (IFN-γ and IL-2) in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. T cells (2×10⁵) were isolated from the splenocytes of C57BL/6JNifdc and B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 13-week-old, n = 3), incubated in the presence of anti-mouse CD3ε antibody (BioXCell, BE0001-1, clone 145-2C11, 2 ug/ml) and anti-mCD28 antibody (BioXCell, BE0015-1, clone 37.51, 5 ug/ml) for 24h, 48h and 72h. IFN-γ and IL-2 productions were then tested using ELISA method.

Subcutaneous tumor growth of B-hPD-L1 B16-F10 cells. B-hPD-L1 B16-F10 cells (1×10⁵) and wild-type B16-F10 cells (2×10⁵) were subcutaneously implanted into B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 7-week-old, n=6). Tumor volume and body weight were measured three times a week. (A) Average tumor volume. (B) Body weight. Volume was expressed in mm³ using the formula: V=0.5 × long diameter × short diameter². Results indicate that B-hPD-L1 B16-F10 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

B-hPD-L1 B16-F10 tumor growth curves from individual mice. B-hPD-L1 B16-F10 cells (1×10⁵) and wild-type B16-F10 cells (2×10⁵) were subcutaneously implanted into B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 7-week-old, n=6). Results indicate that B-hPD-L1 B16-F10 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

PD-L1 expression evaluated on B-hPD-L1 B16-F10 tumor cells by flow cytometry. B-hPD-L1 B16-F10 cells were subcutaneously transplanted into B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 7-week-old, n=6). At the end of the experiment, tumor cells were harvested and assessed for mouse PD-L1(BioLegend, 124312) and human PD-L1(BioLegend, 329706) expression by flow cytometry. As shown, human PD-L1 was highly expressed on the surface of tumor cells. Therefore, B-hPD-L1 B16-F10 cells can be used for in vivo efficacy studies evaluating novel PD-L1 therapeutics.

Subcutaneous tumor growth of B-hPD-L1 MC38 plus cells. B-hPD-L1 MC38 cells (2×10⁵ and 5×10⁵) and wild-type MC38 cells (5×10⁵) were subcutaneously implanted into B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 9-week-old, n=6). Tumor volume and body weight were measured three times a week. (A) Average tumor volume. (B) Body weight. Volume was expressed in mm³ using the formula: V=0.5 × long diameter × short diameter². Results indicate that B-hPD-L1 MC38 plus cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

B-hPD-L1 MC38 plus tumor growth curves from individual mice. B-hPD-L1 MC38 cells (2×10⁵ and 5×10⁵) and wild-type MC38 cells (5×10⁵) were subcutaneously implanted into B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 9-week-old, n=6). Results indicate that B-hPD-L1 MC38 plus cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

PD-L1 expression evaluated on B-hPD-L1 MC38 plus tumor cells by flow cytometry. B-hPD-L1 MC38 plus cells were subcutaneously transplanted into B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 9-week-old, n=6). At the end of the experiment, tumor cells were harvested and assessed for mouse PD-L1 (BioLegend, 124312) and human PD-L1 (BioLegend, 329706) expression by flow cytometry. As shown, human PD-L1 was highly expressed on the surface of tumor cells. Therefore, B-hPD-L1 MC38 plus cells can be used for in vivo efficacy studies evaluating novel PD-L1 therapeutics.

Antitumor activity of IBI363-analog against syngeneic tumors. (A) Tumor growth curves. (B) Body weight changes during treatment. (C) Tumor weight. (D) Lung weight. As shown in panel A, IBI363-analog (provided by the client) was efficacious in controlling tumor growth in B-hPD-1plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice, demonstrating that B-hPD-1plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice provide a powerful preclinical model for in vivo evaluation of PD-1/IL2 bispecific antibodies. The overage of this tumor model is 40%.

Antitumor activity of IBI363-analog and Keytruda-analog against syngeneic tumors. Tumor cells growth of individual mouse. These results demonstrate that B-hPD-1plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice provide a powerful preclinical model for in vivo evaluation of PD-1/IL2 bispecific antibodies

IBI363-analog significantly remodels intratumoral T cell infiltration and effector phenotypes in B-hPD-L1 MC38 plus tumor-bearing B-hPD-1plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. B-hPD-L1 MC38 plus tumor cells were subcutaneously inoculated into B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. Tumor-infiltrating lymphocytes (TILs) were isolated at the end of the study and analyzed by flow cytometry.

Antitumor activity of IBI363-analog and Keytruda analog against syngeneic tumors. (A) Tumor growth curves. (B) Body weight changes during treatment. As shown in panel A, IBI363-analog (provided by the client) was efficacious in controlling tumor growth in B-hPD-1plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice, demonstrating that B-hPD-1plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice provide a powerful preclinical model for in vivo evaluation of PD-1/IL2 bispecific antibodies. The overage of this tumor model is 40%.

Antitumor activity of IBI363-analog and Keytruda-analog against syngeneic tumors. Tumor cells growth of individual mouse. These results demonstrate that B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice provide a powerful preclinical model for in vivo evaluation of PD-1/IL2 bispecific antibodies.

IBI363-analog significantly remodels intratumoral T cell infiltration and effector phenotypes in B-hPD-L1 B16-F10 tumor-bearing B-hPD-1plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. B-hPD-L1 B16-F10 tumor cells were subcutaneously inoculated into B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. Tumor-infiltrating lymphocytes (TILs) were isolated at the end of the study and analyzed by flow cytometry.