The 4-1BB CAR-T Enigma: who’s driving and where can they go?

By Paul Rennert

Introduction to 4-1BB

The 4-1BB receptor (or CD137) is a cell surface protein belonging to the tumor necrosis factor receptor (TNFR) superfamily. 4-1BB is expressed on activated T cells, natural killer cells (NK), dendritic cells (DC), and monocytes. Most attention has focused on 4-1BB-mediated T cell activity, a bit on NK cell and DC activity.

The receptor binds to its ligand, 4-1BBL (CD137L), a trimeric protein expressed on B cells, dendritic cells and macrophages – the canonical antigen-presenting cells (APC). These cell types present foreign antigens derived from diseased or infected cells to T cells. T cell activation upregulates 4-1BB expression, which then binds 4-1BBL. Binding causes the receptor to trimerize and that in turn induces signaling from outside the cell via the 4-1BB extracellular domain to inside the cell, where the 4-1BB cytoplasmic domain functions. The resulting signaling cascade within the cell activates the potent NF-κB and MAPK pathways. These signaling pathways promote T cell survival, proliferation and cytokine production, thereby enhancing T cell persistence and function. Since the function most clearly enhanced by 4-1BB is cytotoxic activity, this pathway has been heavily investigated as an anti-tumor immune pathway. Activating antibodies and purified 4-1BB trimeric proteins were developed to specifically signal through 4-1BB in to induce anti-tumor immunity. Those direct-targeting 4-1BB “agonists” failed in cancer patient clinical trials due to organ toxicities, especially liver toxicity.

Next generation 4-1BB antibodies incorporate a second binding domain to steer 4-1BB activity to tumor antigens (eg. Her2) or to the tumor expressed PD-L1 protein. I wrote recently about the apparent failure of these agonist, bispecific anti-41BB antibodies to mediate productive antitumor immunity in patients (https://shorturl.at/j843m). I proposed that the bispecific antibodies are engaging the wrong kind of second target thus pulling the 4-1BB activity out of an immunologically productive niche and into tumor microenvironments. If true, this hypothesis predicts that whatever 4-1BB activity was triggered in the TME was not physiologically relevant or useful for the T cells.

On the other hand, we know we can take the 4-1BB signaling domain out of its normal milieu and use it to activate T cells when it is linked to an engineered cell surface “chimeric antigen receptor” (CAR) domain. CAR domains use a patient’s own T cells to target a protein on a tumor cell, triggering the T cell to kill the tumor cell. This strategy works well in many Non-Hodgkin Lymphoma (NHL) and Multiple Myeloma (MM) patients, but not nearly as well in solid tumors like glioblastoma, colon cancer or pancreatic cancer.

Incorporating 4-1BB signaling into CAR-T cells supports anti-tumor activity and these cells show improved expansion and persistence in patients compared to the other widely used costimulatory domain from the CD28 protein. A quick reminder here that CD28 is the T cell’s necessary and sufficient “signal two” for activation, after “signal one” whereby the T cell binds to an APC, activating the T Cell Receptor (TCR) and its’ subunit CD3z. CD28 is then engaged by the APC-expressed proteins B7-1 and B7-2, aka CD80 and CD86. The activation cascade involves both T cell and B cells, and initially occurs in specialized lymph node niches called germinal centers. These lymphocytes can then move to the site of activation, whether that is a tumor, an infection or a simple scratch on the arm.

Notably, the cascade involves discreet steps: 1) The T cell is activated by encountering antigen displayed by an APC cell binding the TCR and the TCR subunit, CD3z, 2) CD28 is bound by a B7 protein, 3) the cytokines IL-2 and TNF are secreted by the T cell and several TNFR-family proteins, including 4-1BB, are upregulated, 4) the APC expresses the TNFR protein CD40, and also expressed the 4-1BBL, while the T cell upregulates CD40L, 5) adhesion proteins on T cells and APC cells engage, promoting extended T cell:APC interaction via the adhesion pairs ICAM:LFA1 and CD2:CD58. It’s complicated and extensively regulated so that only T and APC cells that are recognizing foreign antigens (such as expressed by infected cells or tumor cells) become productively activated. The system is most famously counter-regulated by CTLA4, PD-1 and PD-L1, immune checkpoint pathways that block T cell interactions with APC and have been exploited pharmacologically (eg. using ipilimumab, nivolumab, pembrolizumab, or atezolizumab).

When this system is perturbed, the activated T and B cells may die, or worse, may go on to cause pathologies such autoimmune disease and chronic inflammation or, conversely, immunodeficiency disorders. The development of CAR-T cells is therefore somewhat surprising. Here we have a technology that artificially combines the cell surface CAR domain with intracellular signaling domains. As noted above, the two most widely used combinations of signaling domains are CD28/CD3z and 4-1BB/CD3z. Ripped from the normal regulatory constraints of TCR-based ligand binding and the elegant cascade of signals seen on normal T cells, these CAR constructs can induce cytokine secretion, T cell proliferation and cytotoxic activity. So, what happened to the elegant series of activation steps and their careful regulation? What can we learn from these artificial systems?

The CD28/CD3z combination appears straightforward since both signal 1 (CD3z) and signal 2 (CD28) are represented in the CAR signaling domain. This CAR design is represented by the CD19-directed CAR-T therapy Yescarta, aka axi-cel. Axi-cel is used in the treatment of high-risk and relapsed or refractory (r/r) NHL (B cell lymphoma) patients. The CAR-T cells proliferate, expand and kill lymphoma cells. The 4-1BB/CD3z CAR design is used in Kymriah (tisa-cel) and Breyanzi (liso-cel) for r/rNHL, and Carvetki (cilta-cel) and Abcema (ide-cel) for r/rMM. These CAR-T cells also proliferate, expand and kill lymphoma cells, although it’s fair to say that the anti-NHL CAR-T cells are more effective than the anti-MM CAR-T cells. Also, the kinetics of the CAR-T cell populations vary.

One question we can ask, especially as we consider the stepwise and highly regulated T cell signaling cascade, is “how do these 4-1BB/CD28 CARs skip over the CD28 activation step?” Not only is that step missing, but old 4-1BB knockout experiments show that, unlike CD28, 4-1BB is neither necessary nor sufficient for T cell activation. What gives?

A recent paper uploaded to BioRxiv explores the role of endogenous CD28 signaling in the support of CAR-T cell activity (https://doi.org/10.1101/2024.03.21.586084). The CAR construct incorporated 4-1BB and CD3z signaling domains. The tumor targeting domain on the CAR-T cell surface was directed to the multiple myeloma antigen BCMA. The investigators use several techniques to explore the role of CD28, naturally expressed on the CAR-T cells, in support of the 4-1BB/CD3z CAR activities. Using inducible knockout technology as well as antagonist CD28 biologics they showed that CD28 signaling was required for optimal adaptation of CAR-T function in the bone marrow tumor microenvironment (BME; MM cells preferentially reside in the bone marrow). Specifically, it was shown that CD28 modulated T cell metabolic activity and that CD28 signaling was required for the sustained in vivo function of 4-1BB/CD3z CAR T cells. The authors note that the NHL, MM and B cell leukemia tumors targeted clinically by CAR-T cells express CD80, CD86 or both. Therefore, for the CAR-T therapies targeting CD19 and BCMA, CD28 signaling is abundantly supported.

What about the solid tumor setting? Several papers have specifically identified CD28 co-stimulation in both the LN and the TME as critical for anti-tumor cytotoxic T cell differentiation and function. I’ve often written about the critical role of lymph nodes and tertiary lymphoid organs in anti-tumor immunity, and this is a niche in which the CD28 ligands B7-1 and B7-2 would be expected. However, Olejniczak and colleagues correctly question whether the context in which endogenous CD28 encounters ligands, and then signals, impacts the anti-tumor efficacy of the 4-1BB co-stimulated CAR. Simply put, 4-1BB CARs may work terrifically well in the context of NHL and MM where the antigens (CD19 and BCMA, respectively) are co-expressed with B7-ligands that can bind CD28.

In contrast, even if there is CD28 expressed in the solid tumor microenvironment, on APCs for example, it is hard to envision how that signal would integrate with a signal to the CAR binding to an antigen on the solid tumor cell. Indeed, if coordinated signaling is a critical aspect of normal T cell activation then that coordination may be missing in the TME setting.

There are of course reports that 4-1BB signaling enhances the activity of CD28-based CAR-T cells, as would be expected. Indeed, 3rd generation CAR-T constructs include both CD28 and 4-1BB domains. Several refinements include co-expression of these two domains in cis, and mutations within one domain or the other. There is at present no clear consensus on which costimulatory sequence or sequences is best, and it seems likely that this will depend on the antigen targeted and other variables. My best guess is that the best current construct has a 4-1BB costimulatory domain paired with a source of CD28 activity.

In the context of Aleta Bio’s CAR T Engager (CTE) technology we have built diverse functionalities into the CTEs, including costimulatory factors, immune checkpoint antagonists and stimulatory cytokines. In the solid tumor setting several of these functions will likely be required. I can envision building CAR/CTE combinations that can encompass multiple beneficial functions, without the need for extensive CAR-T genetic editing except to secrete the small CTE proteins.

Stay tuned.