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Molecular Cancer Therapeutics
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Cancer Biology and Translational Studies

TSR-033, a Novel Therapeutic Antibody Targeting LAG-3, Enhances T-Cell Function and the Activity of PD-1 Blockade In Vitro and In Vivo

Srimoyee Ghosh, Geeta Sharma, Jon Travers, Sujatha Kumar, Justin Choi, H. Toni Jun, Marilyn Kehry, Sridhar Ramaswamy and David Jenkins
Srimoyee Ghosh
1TESARO, Inc., Waltham, Massachusetts.
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  • For correspondence: sghosh@tesarobio.com
Geeta Sharma
1TESARO, Inc., Waltham, Massachusetts.
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Jon Travers
1TESARO, Inc., Waltham, Massachusetts.
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Sujatha Kumar
1TESARO, Inc., Waltham, Massachusetts.
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Justin Choi
2AnaptysBio, San Diego, California.
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H. Toni Jun
2AnaptysBio, San Diego, California.
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Marilyn Kehry
2AnaptysBio, San Diego, California.
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Sridhar Ramaswamy
1TESARO, Inc., Waltham, Massachusetts.
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David Jenkins
1TESARO, Inc., Waltham, Massachusetts.
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DOI: 10.1158/1535-7163.MCT-18-0836 Published March 2019
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    Figure 1.

    Simultaneous blockade of LAG-3 and PD-1 synergizes to elicit increased efficacy, immune stimulation, and immunologic memory in mouse A20 lymphoma model. A, A20 cells were implanted subcutaneously into Balb/c mice, and tumors were grown to 30 to 50 mm3 before randomization (n = 10 per group) for treatment with either isotype control, anti-LAG3, anti-PD-1, or the combination (10 mg/kg, twice weekly; Materials and Methods); coefficient of drug interaction, CDI <0.7 (significant synergy). B, Mice (n = 4 per group) were sacrificed on day 36, and pharmacodynamic changes in immune cells in the spleen were assessed. The combination group displayed a significant increase in proliferating T cells and total CD8 T cells, relative to anti-PD-1 treatment alone. C, Surviving animals in each group were monitored for tumor-free survival for 40 days, followed by rechallenge with A20 cells (n = 4 and n = 6, for anti-PD-1 and combination treatment groups, respectively). Naïve mice (n = 6) were inoculated in parallel and monitored for tumor growth. D, Significant increase in effector-memory (CD44+CD62L−) T cells in the combination group and higher proportion of splenic IFNγ+ CD8 T cells, compared with PD-1 blockade alone. Mice were sacrificed on day 41 post-rechallenge and spleens assessed for immune cell populations by flow cytometry; *, P ≤ 0.05; **, P ≤ 0.01, unpaired Student T test. E, Kaplan–Meier survival curves for treatment arms; **, P ≤ 0.01 by the log-rank (Mantel–Cox) test, relative to isotype control.

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    Figure 2.

    TSR-033 binds with high affinity to human and cyno LAG-3. A, TSR-033 binds human and cyno LAG-3 expressed on the surface of CHO-S cells, but not mouse LAG-3, as assessed by flow cytometry. B, TSR-033 binds LAG-3 on resting CD4 and CD8 T cells in health human donors (n = 3); total LAG-3 was detected using a noncompeting antihuman LAG-3 antibody (Materials and Methods). C, TSR-033 inhibits binding of labeled LAG-3 Fc fusion protein to MHC Class II expressing Daudi cells. D, TSR-033 relieves LAG-3–mediated repression of an NFAT-responsive element, downstream of TCR signaling. Data are representative of at least two separate occasions.

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    Figure 3.

    TSR-033 amplifies T-cell effector function, particularly in combination with anti-PD-1. The functional activity of TSR-033 was evaluated in an MLR assay, in which primary human CD4 T cells were mixed with monocyte-derived dendritic cells from a different donor. In these studies, dendritic cells and allogeneic CD4 T cells were incubated (A) in the presence of TSR-033 or isotype control or (B) TSR-033, TSR-042, and isotype control at indicated concentrations for 48 hours, and activation of T cells was determined by measuring the level of IL2 secretion. C, The ability of TSR-033 and TSR-042 to induce IL2 from human PBMCs (n = 5 donors) stimulated with 100 ng/mL SEB for 3 days was assessed. Data are representative of at least two separate occasions.

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    Figure 4.

    Dual blockade of LAG-3 and PD-1 using TSR-033 and TSR-042 improves therapeutic efficacy and T-cell stimulation in a humanized NSCLC tumor mouse model. A, The combination of TSR-033 with TSR-042 has an additive effect (CDI = 1.001) on restricting tumor growth in HuNOG-EXL mice inoculated with A549 cells. Mice were randomized at tumor volumes of 80 to 120 mm3, followed by administration of the indicated regimens (Materials and Methods). Tumor growth inhibition at termination for each treatment arm is indicated in parentheses. B, Relative to TSR-042 monotherapy, the combination group had increased intratumoral T cells and proliferating CD8 T cells; *, P ≤ 0.05, unpaired Student T test. C, The combination of TSR-033 with TSR-042, compared with TSR-042 monotherapy, resulted in significant reduction in TAMs, M2 TAMs and increased M1/M2 ratios. TAMs were identified as CD45+CD3−CD20−CD68+, M2 TAMS, CD45+CD3−CD20−CD68+HLA-DRloCD209+, and M1 TAMs, CD45+CD3−CD20−CD68+HLA-DRhiCD209−. Data represent two independent experiments (n = 10 per treatment arm) and have been normalized to fold change over isotype control for each treatment arm in B; *, P ≤ 0.05, unpaired Student T test.

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    Figure 5.

    Increased effector-memory T cells and ex vivo cytokine production by splenic T cells from the combination group, relative to TSR-042 alone in a humanized NSCLC tumor mouse model. A, Increase in proliferating and effector-memory (CD45RA−CCR7−) CD4 and CD8 T cells in the combination group compared with TSR-042 alone. B, Significant increase in IFNγ and TNFα production by CD4 T cells in the combination group over TSR-042 alone, upon ex vivo stimulation of mouse splenocytes (Materials and Methods); *, P ≤ 0.05, unpaired Student T test.

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    Figure 6.

    Secondary upregulation of LAG-3 following PD-1 blockade. A, Significant increase in LAG-3 expression on T cells within A549 tumors and spleens of humanized NOG-EXL mice following TSR-042 treatment, relative to isotype control (end of study). Significant reduction in (B) IL2 production but not (C) IFNγ production by intratumoral and splenic LAG-3–positive T cells in both the isotype control and TSR-042 groups; *, P ≤ 0.05; **, P ≤ 0.01, unpaired Student T test.

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  • Table 1.

    TSR-033 binding to LAG-3 by surface plasmon resonance (SPR) and LAG-expressing CHO cells

    Kinetic parameters (SPR)aLAG-3–expressing CHO cellsb
    SpeciesKassoc (milliseconds)−1Kdissoc (seconds)−1KD (nmol/L)EC50 (nmol/L)EC90 (nmol/L)
    Human LAG-31.3 × 1051.25 × 10−40.950.811.9
    Cynomolgus LAG-31.1 × 1052.3 × 10−42.02.668.5
    • ↵aKassoc, association rate constant; Kdissoc, dissociation rate constant; KD, dissociation constant.

    • ↵bTSR-033 binding not detected on nontransfected CHO cells.

Additional Files

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  • Supplementary Data

    • Supplementary Table 1 - Comparison of mouse and humanized anti-PD-1 and anti-LAG-3 antibodies used in syngeneic and humanized mouse tumor models
    • Supplementary Table 2 - A Bio-Layer Interferometry (BLI)-based method (Forte Bio Octet Red 96) for determining binding rate constants (kon, koff, KD) was utilized to provide kinetic data on the interaction between the monoclonal antibodies (human IgG1 reference standard and TSR-033) and human CD16a
    • Supplementary Fig 1 - Profiling of human T, myeloid and NK cells in the spleens of HuNOG-EXL mice (n=3) at day 10 post-inoculation with the A549 cell line
    • Supplementary Fig 2 - TSR-033 binding to LAG-3 by Surface Plasmon Resonance (SPR)
    • Supplementary Fig 3 - Kaplan-Meier survival curves for huNOG-EXL mice implanted with A549 NSCLC cells treated with isotype control, TSR-042, TSR-033 or TSR-042+TSR-033
    • Supplementary Fig 4 - TSR-033 shows little to no binding to C1q by ELISA relative to a human IgG1 reference standard
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Molecular Cancer Therapeutics: 18 (3)
March 2019
Volume 18, Issue 3
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TSR-033, a Novel Therapeutic Antibody Targeting LAG-3, Enhances T-Cell Function and the Activity of PD-1 Blockade In Vitro and In Vivo
Srimoyee Ghosh, Geeta Sharma, Jon Travers, Sujatha Kumar, Justin Choi, H. Toni Jun, Marilyn Kehry, Sridhar Ramaswamy and David Jenkins
Mol Cancer Ther March 1 2019 (18) (3) 632-641; DOI: 10.1158/1535-7163.MCT-18-0836

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TSR-033, a Novel Therapeutic Antibody Targeting LAG-3, Enhances T-Cell Function and the Activity of PD-1 Blockade In Vitro and In Vivo
Srimoyee Ghosh, Geeta Sharma, Jon Travers, Sujatha Kumar, Justin Choi, H. Toni Jun, Marilyn Kehry, Sridhar Ramaswamy and David Jenkins
Mol Cancer Ther March 1 2019 (18) (3) 632-641; DOI: 10.1158/1535-7163.MCT-18-0836
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