Intracellular Immune Checkpoints - SHP-2, CBL-b, CISH, HPK1

Immune checkpoints are negative feedback mechanisms of the immune system that, when activated, protect normal cells from T cell-mediated cytotoxicity and maintain immune homeostasis. Well-known checkpoints (PD-1, CTLA-4, TIM3, LAG3, etc.) are expressed on the surface of the cell membrane. In addition to this, a number of intracellular proteins are involved in negative feedback loops downstream of the TCR and can be considered as intracellular immune checkpoints, several of which are currently in the clinical research stage.

Creative Biolabs introduces some intracellular immune checkpoints and gives some relevant information about their development and application.

Src Homology Region 2 (SHP-2)

PD-1 inhibits T cell activation by recruiting the phosphatase SHP-2. However, T cell-specific deletion of SHP2 in mice did not improve anti-tumor immunity. It was later found that in the absence of SHP-2, PD-1 recruited SHP-1 to maintain this function. Thus, blocking both SHP-1 and SHP-2 is necessary to improve the TCR signaling pathway.

SHP-2 and redundant mechanisms in T cell inhibitory receptor signaling. (Niogret, Charlène, et al., 2019)Fig. 1 SHP-2 and redundant mechanisms in T cell inhibitory receptor signaling.1

In the bone marrow, GM-CSF induces phosphorylation of PD-1 and recruitment of PD-1-SHP-2 to the GM-CSF receptor. Deletion of SHP-2 or PD-1 enhances GM-CSF-mediated phosphorylation of the transcription factors HOXA10 and IRF8, which regulate medullary differentiation and mono-moDC spectrum differentiation, respectively.

  • SHP-2 and PD1-SHP-2 signaling inhibits myeloid differentiation, generates suppressive myeloid lineage cells, and suppresses antitumor immunity.
  • SHP-2 is an important regulatory molecule of the growth factor-ERK signaling pathway.

There are many ongoing clinical studies of anti-PD-1 antibodies in combination with SHP-2 inhibitors, including:

  • SHP2 inhibitor BBP-398 in combination with Nivolumab in patients with advanced non-small cell lung cancer with a KRAS mutation
  • A dose escalation study of SHP2 inhibitor in patients with solid tumors
  • Harboring SHP2 inhibitor ET0038 monotherapy in patients with advanced solid tumors

Casitas B-lymphoma b (CBL-b)

CBL proteins have a ring-finger catalytic structural domain that is responsible for protein ubiquitination and sequential degradation of target proteins. CBL proteins lead to the degradation of multiple targets, thereby down-regulating the TCR signaling cascade. CBL-b targets the regulatory subunit of PI3K, p85, which reduces the activity of PI3K and interferes with its ability to activate different signaling pathways.

CBL-b is involved in co-stimulatory signaling that regulates CD28 or inhibits the receptors CTLA-4 and PD-1. Loss of CBL-b on the TCR leads to Akt/Erk phosphorylation, proliferation, activation, cytokine production (IFN γ, TNFα, IL-2), and cytolytic capacity (granzyme B).

Depiction of intracellular Cbl-b signaling in the tumor microenvironment. (Augustin, Ryan C., et al., 2023)Fig. 2 Depiction of intracellular Cbl-b signaling in the tumor microenvironment.2

Several clinical trials to inhibit CBL-b in T cells are ongoing.

  • Computational approach using DEL to identify novel, small molecule CBL-b inhibitors
  • Enhanced cell therapy via CD79a/CD20 CAR-T with CBL-b knockout


CISH, a member of the Suppressor of Cytokine Signaling (SOCS) family of proteins, negatively regulates the CD8+ T cell signaling pathway. CD8+ T lymphocytes from defective mice showed improved response to TCR signaling and increased expression of effector function-related genes (Il2, Prf1, GrzmB, Eomes, Tbx21, c-Myc, and Bcl2l).

Clinical trials are underway to knock down CISH in TILs by CRISPR-Cas9 for tumor therapy.

Hematopoietic Progenitor Kinase 1 (HPK1)

HPK1, encoded by the MAP4K1 gene, is a protein kinase identified as a key regulator of TCR signaling. HPK1 is activated by the TCR complex, and HPK1 binds to and phosphorylates SLP-76 on the LAT signaling vesicle. Phosphorylated SLP-76 subsequently binds to GAD and 14-3-3 proteins, which disrupt SLP-76's interaction with the LAT signalosome, thereby triggering SLP-76 degradation. Interaction with LAT signalosome, thereby triggering SLP-76 degradation, which negatively regulates MAPK-ERK pathway signaling.

HPK1 knockout in mice results in increased T cell proliferation, activation, and cytokine secretion, thereby conferring the ability to control tumor growth. Many HPK1 small molecule inhibitors are undergoing clinical trials for cancer immunotherapy.

At Creative Biolabs, we are at the forefront of these advances, working to immune checkpoint targeted small molecule development. Please do not hesitate to contact us with your particular needs.


  1. Niogret, Charlène, et al. "SHP-2 in lymphocytes' cytokine and inhibitory receptor signaling." Frontiers in immunology 10 (2019): 2468.
  2. Augustin, Ryan C., et al. "Targeting Cbl-b in cancer immunotherapy." Journal for Immunotherapy of Cancer 11.2 (2023).

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