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.

Recent CAR-T Clinical Studies in Glioma and Glioblastoma Patients

Three exciting recent papers have hinted at the promise of CAR-T therapy in the setting of aggressive CNS malignancy, but also reveal critical issues limiting success.

Author: Paul D Rennert, President & CSO, Aleta Biotherapeutics

A brief introduction

Glioblastoma (GBM) and high-grade glioma (HGG) are fast-growing and deadly tumors that spread within normal brain tissue and are therefore difficult to eradicate. Surgery, chemotherapy and radiation therapy are the standard treatment options, sometimes paired with anti-VEGF antibody (bevacizumab) therapy, but these regimens all fail to produce durable responses for patients. Median OS for GBM at first recurrence ranges from 5.5 to 12.6 months and post-bevacizumab is between 3 and 4 months. Thus, patients progress rapidly after therapy and experience poor quality of life prior to eventual death. The unmet need for patients with these cancers is extreme.

Chimeric antigen receptor T cells (CAR-T) are directed to attack tumor cells via recognition of tumor antigens and have been remarkably successful in the treatment of B cell cancers including leukemias, lymphomas and myeloma. In the following papers CAR-Ts were targeted to CNS tumor antigens in order to evaluate safety, preliminary efficacy and biomarkers of therapy such as CAR-T cell persistence and distribution in the patients and the induction of pro-inflammatory cytokines associated with anti-tumor immunity, such as interferon-gamma (IFNg).

Three interesting papers

Following is a brief discussion of the papers in which unique characteristics of each CAR and T cell technology are considered in light of the reported outcomes. The investigators used a variety of tumor targeting strategies and CAR-T manufacturing techniques (Table 1) and different CAR-T delivery methods and doses (Table 2). All CAR vectors used 4-1BB and CD3z signaling domains to trigger cell activation.

1 - Choi et al (2024). Intraventricular CARv3-TEAM-E T Cells in Recurrent Glioblastoma. NEJM. DOI: 10.1056/NEJMoa2314390. (from Mass General Hospital)

Choi et al. have opened their clinical study with three recurrent GBM patients. GBM is a cancer whose cells that rely on growth factor EGFR signaling for proliferation and survival signal, indeed many GBM patients have EGFR gene amplification and/or have mutations in the EGFR sequence that support constant positive signaling.

Patients were treated with CAR-T cells engineered to recognize EGFRvIII which is an EGFR splice variant expressed by tumor cells. The CAR-T cells were also engineered to secrete a bispecific T cell engager protein (BiTE) that recognizes the normal form of EGFR (the BiTE is an anti-CD3/anti-EGFR engineered from antibody sequences). CAR-T cells were delivered to the patients in a single intraventricular infusion (ICV, see Table 2). Of note, analysis of the infused cells showed that the BiTE protein bound to the CAR-T cells, thus 93% of the CAR-T cells were preloaded with the BiTE (their Figure 4b). It follows that each CAR-T cell initially could be activated in two ways: through the CAR domain upon binding to EGFRvIII and through the preloaded BiTE upon binding to EGFR. It is unclear if the BiTE alone also activated normal CD3-positive T cells in the patients although this is theoretically possible.

Treatment with CARv3-TEAM-E T cells did not cause any severe adverse events and no dose-limiting toxicity was observed in these 3 patients. Tumor regression occurred rapidly post-CAR-T infusion and one patient had a durable response through 5 months as measured by MRI and by measurement of EGFR and EGFRvIII transcripts in the cerebrospinal fluid (CSF).

Quantification of CAR-T cells and BiTE-loaded CAR-T cells showed transient cell persistence in CSF samples with rapid decline through day 28 (see their Figure 4C). Patient 2 - who obtained the longest response - had the lowest expansion in the CSF of both CAR-T cells and of BiTE-loaded CAR-T cells (see Figure 4C in their paper) but had the highest measured expression of IFNg that is expressed by T cells, including CAR-T cells, when they are activated. Thus, presence of the CAR-Ts and the secreted BiTE in the CSF was not correlated with clinical response in this small study.

In the follow-up, this study will enroll recurrent and newly diagnosed GBM patients for a multi-dose regiment of CARs given weekly x 6 weeks (NCT05660369).

2 - Bagley et al. 2024. Intrathecal bivalent CAR T cells targeting EGFR and IL13Rα2 in recurrent glioblastoma: phase 1 trial interim results. Nat. Med. https://doi.org/10.1038/s41591-024-02893-z  (University of Pennsylvania)

Bagley and colleagues treated 6 refractory GBM patients with intrathecally (IT) delivered bispecific CAR-T-cells targeting EGFR epitope 806 (specific for tumor cells) and IL13Rα2, a known GBM antigen (Table 1). The use of binders to two different antigens is to address the known problem of tumor antigen heterogeneity leading to relapse as has been seen when only IL13Rα2 was targeted. EGFR epitope 806 is known to be expressed on 50–60% of GBM patient tumor cells and IL13Rα2 is known to be expressed on 50–75% of GBM patient tumor cells. Therefore, one or both of the antigens is expected to be present on nearly all GBM tumor cells.

As in the MGH study, reductions in MRI enhancement and measured tumor size were observed in all patients at early imaging timepoints although none met the criteria for clinical response (tumor volume reduced > 30%). Tumor size was reduced on the first MRI scan obtained 24–48 h after CAR T cell administration, and partial tumor regression maintained at day 28+ in a subset of patients including one patient with stable disease beyond 4 months post-CAR treatment.

Using biomarker analyses similar to the prior study, CAR-T cells and inflammatory cytokines were detected in the CSF of all patients. Peak CAR-T cellularity in the CSF was observed between days 1 and 7 post-CAR, reaching an average of 109,235 copies of CAR per ug of genomic DNA. Note these levels are 2x-10x higher than that obtained in the MGH study. Possibly because such levels were achieved, CAR-T cells were also found in the peripheral blood in all patients.

Resistance mechanisms thought to limit anti-GBM immunity include antigen loss, as discussed above, a highly immunosuppressive tumor microenvironment (TME) and T cell dysfunction of the CAR-T cells in the infused product or within the TME. In line with the next study, the UPenn group plans to characterize CAR-T cell effector/memory phenotypes prior to infusion to look for correlations with clinical outcomes (NCT05168423).

3 - Brown et al (2024). Locoregional delivery of IL-13Rα2-targeting CAR-T cells in recurrent high-grade glioma: a phase 1 trial. Nat Med. https://doi.org/10.1038/s41591-024-02875-1 (City of Hope)

The City of Hope team enrolled 65 patients with recurrent HGG, the majority of which had recurrent GBM. 58 patients were evaluable.

The study tested 3 routes of T cell administration: intratumoral (ICT), intraventricular (ICV) and dual (ICT/ICV). Arm 5 of the study used dual ICT/ICV delivery and an optimized manufacturing process that allowed expansion to the clinical maximum feasible dose of 2 × 108 cells. Arm 5 CAR-T cells were CD62L+ enriched naive, stem cell memory and central memory T cells (Tn/mem). Evaluable patients received at least 3 weekly infusions of CAR-T cells and additional infusions were allowed until disease progression. Table 2 summarizes these details.

Stable disease or better was achieved in 50% (29/58) of patients, with two partial responses (PR: >30% reduction in tumor diameter) one complete response (CR: 100% reduction in tumor size) and a second CR after additional CAR-T cycles off protocol. Focusing on Arm 5 that used optimized CAR-T cell manufacturing and Tn/mem cells, 9/21 patients (43%) survived beyond 12 months and median OS was 10.2 months.

CAR-T cells were detected in the CSF in the most patients 1 day post infusion for at least one cycle, and for a subset of patients, ≥7 days post infusion, reaching a maximum of ~700 copies per ug of DNA in arm 5, albeit with very large error bars (see their Figure 4b). CAR-T cells were also detected in peripheral blood, reaching the highest concentration in arm 5 patients. Notably, CD3+ T cell density measured in tumor biopsy samples pre-CAR infusion correlated with median OS, suggesting a pre-existing anti-tumor immune response. Finally, this study demonstrated that the CAR-T cells delivered to the CNS can persist in the CSF and traffic to the periphery (see the Discussion, below), similar to the report from University of Pennsylvania (UPenn).

This trial will move forward using Arm 5 processes (Tn/ mem manufacturing and dual ICT/ICV delivery) in three declared studies, one with or without nivolumab (anti-PD-1 antibody) and ipilimumab (anti-CTLA4 antibody) to counter immunosuppression (NCT04003649), one for pediatric patients (NCT04510051) and one for leptomeningeal GBM, ependymoma, or medulloblastoma patients (NCT04661384).

A brief discussion

What we see here are signs of promise in the development of CAR-T therapy for a deadly solid tumor type. Each study presents 1 or more patients in which a number of positive signals are detected: reduction in tumor volume, expansion of CAR-T cells, induction of IFNg and other pro-inflammatory cytokines, and in the final study by Brown et al., association with CD3 T cell infiltration of the tumor itself. The challenges encountered are consistent with current views regarding antigen-loss relapse and the immunosuppressive nature of the GBM TME.

The story acquires a little more depth as we look across the studies.

In the first paper from MGH, two different anti-EGFR targeting strategies are used, one to target wildtype (ie. normal) EGFR, and one to target a splice variant EGFRvIII that is expressed only by tumor cells. Two modalities are also used, the CAR-T cell itself (anti-EGFRvIII) and the secreted BiTE, an anti-CD3/anti-EGFR protein that activates CAR-T cells by binding to CD3 and binding to the tumor cell-expressed EGFR. It will be interesting to know if the BiTE can activate CD3+ T cells within the patient in addition to activating the CAR-T cells. Dual antigen-targeting should prevent loss of CAR response due to loss of antigen, nonetheless, CAR persistence in this study was limited, as were the anti-tumor responses. Patient 1 lost EGFRvIII expression and temporarily lost EGFR expression and patients 2 and 3 eventually lost expression of both antigens. In this small study it is difficult to draw any correlations between biomarkers and responses.

The UPenn study is another dual antigen-targeting study, using a bispecific CAR to attack the EGFRVIII epitope 806 and antigen IL-13Ra2. They enrolled 6 patients and triggered robust CAR-T cell expansion that peaked at day 7 and was still measurable at day 28 in the CSF and in peripheral blood samples. Despite the robust CAR expansion and concomitant IFNg secretion, anti-tumor responses as measured by MRI were limited in extent to stable disease (SD).

Finally, the City of Hope paper reports on the largest number of patients, while using a variety of strategies. In Arm 5, the use of a selected CAR T phenotype (Tn/mem) was combined with two different means of CAR delivery, and perhaps most critically, a multiple dosing paradigm with at least 3 weekly doses given at the outset.

This paper presents several interesting biomarker observations. CAR persistence was limited after each cycle, at ~ 7 days, and not markedly higher in the CSF of patients in Arm 5 as compared to the other arms in the study. IFNg-related cytokines (signature: IFNg, CXCL9 and CXCL10) were markedly higher in the CSF of Arm 5 patients. Also Arm 5 patients showed the most robust CAR cellularity and IFNg-related signature in the peripheral blood. Finally CD3-positive cellularity in the biopsy samples of patients was correlated with responsiveness regardless of arm of therapy – there were 14 CD3-intermediate and only 3 CD3-high patients represented – but they presented the best survival probability (see their Figure 4A,B).

What can we make of these observations?

IFNg is a cytokine that is directly cytotoxic to tumor cells and plays an outsized role in anti-tumor immunity. CXCL9 and CXCL10 expression is induced by IFNg stimulation of mainly non-immune cells like fibroblasts and epithelial cells but also tumor cells. These chemotactic cytokines, or chemokines, regulate immune cell migration, differentiation, and activation. Immune reactivity occurs through recruitment of immune cells including T cells. Tumor-infiltrating T cells (whether normal T cells or engineered CAR-T cells) are critical for clinical response – this just means that T cells have to get into the tumor to initiate killing. This recruitment can be induced by CXCL9 and CXCL10 that bind to receptors expressed on the T cell surface. These same chemokines play similar roles organizing the distribution of immune cells in immune organs such as lymph nodes and spleen.

Some negative biomarker conclusions: 1) Across these 3 studies CSF CAR-T cellularity seems of limited use in predicting clinical response, although there may be useful data within Arm 5 of the City of Hope study, if it were broken out by patient. 2) CAR-T persistence in all studies is limited. 3) Multi-antigen targeting does not appear to solve the persistence issue at least in these HGG and GBM cancers.

Some positive biomarker conclusions: 1) CAR-T cell response is associated with pre-existing anti-tumor immunity as represented by normal CD3+ T cells within the tumor as measured in biopsy samples pre-CAR-T infusion. 2) CAR-T response is associated with a IFNg signature that includes the T cell-attracting chemokines CXCL9 and CXCL10.

Overall conclusions: an intriguing array of technologies are on display in these papers targeting these intractable and deadly solid tumors. It is tempting to conclude that deep and durable responses may soon be achieved for these patients, but before this can happen the CAR-T persistence issue needs to be solved. Looking ahead, the confluence of diverse technologies will have to continue, and with this in mind I’d look to several areas to contribute: the CAR vaccine technologies as represented by BioNTech, Elicio and many others, the ‘armored’ CARs that can produce additional cytokines or chemokines to broaden the immune attack, the leveraging of bona fide immune cell interactions to improve CAR fitness and persistence - see Seattle Children’s STRIVE-01 and STRIVE-02 trials, Gracell’s BCMA/CD19 dual CAR-T GC012F and the CAR-T Engager technology developed by Aleta Biotherapeutics (www.aletabio.com).

Table 1 presents some of the technical details associated with the CAR genes and the patient T cells.

Table 2 summarizes dosing and clinical details from the 3 papers.

The PD-1/PD-L1 Pathway and CAR-T Cells: Stop Making Sense

(Modified from an essay originally published by Evaluate Vantage)

Author: Paul D Rennert, CEO & CSO, Aleta Biotherapeutics

One of the 16 songs included in the 1984 Talking Heads movie Stop Making Sense is Crosseyed and Painless, with it’s deep funk vibe under alternating soothing and staccato vocals. The song is a masterpiece of the ‘80s alt-rock era; the album Remain in Light is a timeless classic.

This live version from Rome 1980, featuring Adrian Belew on lead guitar, is excellent: youtube.

The song includes this spitting refrain:

I'm ready to leave

I push the fact in front of me

Facts lost

Facts are never what they seem to be

I thought of this lyric the other day when I read about a clinical study presented at the recent EBMT-EHA CAR T meeting. Entitled Pembrolizumab After CAR T-Cell therapy: a Single Center Experience, a study in Diffuse Large B Cell Lymphoma (DLBCL). The presentation was summarized in Blood Cancers Today as follows:

“Since approximately 30%-40% of patients with R/R DLBCL achieve durable remissions, many patients will require subsequent treatments… which is why pembrolizumab is sometimes administered with the hope that it will reverse T-cell exhaustion following CAR T-cell therapy. The study included 59 patients with R/R DLBCL who received commercial CAR-T cell therapy… 31 patients (52.5%) experienced relapse or disease progression after the CAR-T treatment… 17 then received intravenous pembrolizumab 200 mg every three weeks as salvage therapy. The median time from CAR T-cell infusion to the first pembrolizumab treatment was 42 days (range, 16-211) and with a median of two doses administered (range, 1-22)… pembrolizumab was well tolerated. The best overall response rate after pembrolizumab was 23.5% (n=4), with all four of those patients achieving a complete response (23.5%).”(link)

This is an interesting study and I’d like to see more detailed data when available. But there a few key items here to consider. First, the investigators report time since CAR-T infusion, and the range was considerable, from a week and a half to seven months post-CAR. Depending on that timing, and which CAR was administered, CAR-T cellularity in patients will be markedly different at the different time points. It would be useful to see a table that breaks out patient by CAR (axi-cel, tisa-cel), time to first pembro infusion, and response. I’d also like to know the status of the normal B cell and T cell pools in these patients.

The UPenn group published a similar small study (link-1). 12 patients with B-cell lymphomas who were either refractory to (n = 9) or relapsed after (n = 3) treatment with a CD19-directed CAR T-cell therapy (using 4-1BB–costimulation) were given pembrolizumab 200 mg IV every 3 weeks. Median time from CAR T-cell infusion to first pembrolizumab dose was 3.3 months (range, 0.4-42.8 months). Best response after pembrolizumab was 1 complete response and 2 partial responses (ORR=25%).

The UPenn team noted that ““The optimal timing of pembrolizumab is a big question, and it appears that 1 year out is not optimal… Our study suggests that within 3 months is a better time frame, and we may be able to start pembrolizumab much sooner or even before CAR T-cell therapy. We treated some patients as early as 13 days after CAR T-cell infusion without toxicity ...”

So, what in fact is going on with these patients?  We know from many failed clinical studies that immune checkpoint therapy has limited efficacy in NHL. Pembrolizumab is approved for Primary Mediastinal Large B Cell Lymphoma (PMBCL) where a subset of patients respond and some have a durable response (link-2). Anti-PD-(L)-1 therapeutics are also being trialed in combination with SOC (eg. R-CHOP) in the front-line setting, where they have been shown to be safely received. Regardless, progress with immune checkpoints in NHL has been much slower and more limited than in the solid tumor setting and in Hodgkin Lymphoma (HL, a distinct lymphoma from the NHL subtypes).

Note above, in the first quoted paragraph, the statement “pembrolizumab is sometimes administered with the hope that it will reverse T-cell exhaustion following CAR T-cell therapy”. This is a broadly held assumption of how pembrolizumab and other anti-PD-(L)-1 antagonists work, but in this instance, I think the proposed mechanism is potentially misleading. It helps to think about the role the PD-1 pathway plays in normal immune function to understand what is likely happening in the studies referenced above. This subject has been extensively investigated, and reviewed, by many groups including Arlene Sharpe and colleagues (eg. link-3). Simply put, nearly all subsets of activated T cells express PD-1, and these are not all exhausted, in fact most are fully functional. Indeed, PD-1 acts as a T cell activation rheostat, preventing unwanted activation. It is indisputably not a marker of exhausted T cells and in most settings serves to modulate T cell activation as required to mediate immune function without damaging normal cells and driving the T cell into a dysfunctional state of overactivation and exhaustion.

Of course, T cells appear to become functionally exhausted in the setting of chronic antigen presentation, as in chronic viremia and in some solid tumor microenvironments. It is not clear that this happens to CAR T cells within lymphoma patients as both cell populations (target cells and CAR T cells) are rapidly turning over. Biomarker analyses have never identified the PD-1/PD-L1 pathway as a mediator of CAR T response in lymphoma, suggesting that a more complex cellular interplay is taking place. Indeed, one can conceive of cell therapy as a frantic race between the proliferating cancer cells and CAR T cells, both placed under intense selective pressure. The question then is: if PD-1 activity is not hampering the CAR T response to lymphoma by inducing exhaustion, what is happening?

We now know, from analyses performed by Stanford, The Moffitt Cancer Center, Kite/Gilead and others that CD19 antigen density controls CAR T response in lymphoma in the majority of patients (link-4, link-5). The Stanford group has quantified the number of CD19 molecules that need to be expressed on a target cell in order to trigger a CAR response, and this ranges from a few thousand up to tens of thousands, in part based on the costimulatory domain used in the CAR (link-5). Fred Locke (Moffitt) and Rhein Shen (Kite/Gilead) showed last year, that average CD19 ‘brightness’ on lymphoma cells was the single predictor of the effectiveness of CAR T therapy in the 2nd line Zuma-7 DLBCL trial (Locke et al, ASCO22, Shen, IO Summit22).

One can weave these storylines together by asking what CD19 antigen density has to do with response to anti-PD-(L)-1 treatment after CAR relapse. Do we need to invoke ‘exhaustion’ as the de facto explanation for the observation that anti-PD-(L)-1 treatment can reactivate CAR T cells in some patients?

Let’s consider the molecular consequences of signaling through the CAR domain and through PD-1. CAR engagement triggers phosphorylation of the CD3z and costimulatory (CD28, 4-1BB) domains in anti-CD19 CAR T cells. As noted above, a minimum number of contacts between the CD19 protein and the anti-CD19 CAR domain is needed to productively trigger CAR T cell activation, proliferation, and effector functions. While the details differ between 4-1BB based CARs and CD28 based CARs, canonical features such as Lck activation and ZAP70 phosphorylation and the subsequent formation of productive T cell signaling complexes are roughly conserved. Activation of PI3Kinase and its signaling cascade is mediated either directly (CD28-based CAR) or indirectly (4-1BB-based CAR), although many of the details are poorly understood (link-6). As with natural TCR signaling, strength of binding and duration of binding are important variables for productive CAR T engagement (link-7).

PD-1 signaling requires prior T cell activation and is particularly enhanced when CD28 is activated (link-8). Control over this system is mediated in large measure by the recruitment and activation of Lck by T cell activation signals; Lck, as a kinase, phosphorylates downstream signaling proteins but will also phosphorylate PD-1. PD-1 phophorylation drives recruitment of SHP phosphatases, which counter the activation signals. Simplistically, the integration of positive signals from the CAR domains and negative signals from PD-1 occurs at the level of Lck and its signalosome components (link-9, link-10). These pathways are capable of further counter-regulation, as reduction in TCR or CAR signaling reduces PD-1 activity and visa versa.

This model produces a prediction, that the strength of signaling through these two pathways (CAR vs PD-1) controls CAR-T cell responses. This prediction is supported by published analyses of pathway interactions; these can be summarized as finding that PD-1 signaling dramatically shifts the TCR/antigen binding dose–response curve, making T cells much less sensitive to TCR–generated signals (link-11, link-12). Responses to differential TCR strength show that too much or too little TCR signaling are both associated with dysfunction, indicating that the ‘rheostat’ role that PD-1 plays is to keep T cells at an activation state that is ”just right”. In the context of CAR T signaling, the same dynamic appears to be in play, with too much PD-1 signaling occurring in the context of low antigen expression. In the B cell lymphoma setting the appearance of a second mechanism of resistance drives this point home. The TCR complex and the CAR T cell membrane include the protein CD2, which binds to CD58 (aka LFA3) on various cell types including B cells. The CD2/CD58 pathway is not a major mediator of T cell activation except in settings of low antigen density (link-13, link-14). Recent studies of responsiveness to CAR T cell therapy in lymphoma have highlighted loss or downregulation of CD58 expression as a mechanism of tumor cell escape (link-15, link-16). Since the antigen sensitivity of CAR domains is less than that of native TCRs, this route of escape may be deployed within the lymphoma cell population to further restrict the recognition of antigen.

This model provides a simple framework for understanding the mechanisms of resistance to and relapse from anti-CD19 CAR T therapy. As noted previously, Mazner & Mackall analyzed CD19 downregulation in their anti-CD19 CAR T cell treated cohort at Stanford (n=45; link-5). By using quantitative flow cytometry analyses they identified loss or downregulation of CD19 in 63% of their relapsed patients. Rhein Shen of Kite/Gilead presented data at the IO Summit in Boston that showed that the single predictor of lymphoma patient response to anti-CD19 CAR T cell treatment was the brightness of CD19 on the lymphoma cell population. These observations, and the recent data on CD58-downregulation, suggest that the overall density of antigen/CAR interaction determines outcome for most patients; some patients can be “pushed back” to productive CAR signaling by blocking PD-1, thereby changing the balance of positive (CAR) and negative (PD-1) signaling cascades. Of note, there is no need to evoke ‘exhaustion’ in the context of this model. Thus, “Facts are never what they seem to be”.

I’m interested in these questions because Aleta Biotherapeutic’s has developed ALETA-001 a therapeutic that will enter the clinic in September in patients previously treated with an anti-CD19 CAR-T therapy in the UK (the Phase 1/2 trial is sponsored by Cancer Research UK). ALETA-001 is a three-domain biologic that contains the CD19 extracellular domain, an anti-CD20 binding domain and an anti-albumin binding domain. ALETA-001 is designed to coat B cell lymphoma and leukemia cells with CD19 protein bound to CD20 (link-17) and thus changes the cell surface expression of the bound lymphoma cell to be CD19-bright. ALETA-001 added to anti-CD19 CAR therapy will prevent antigen-loss relapse, increases antigen density and enable rapid cytotoxicity at low E:T ratios. We believe that -001 will change outcomes for patients at risk of relapse (as determined at day 28 post-CAR infusion, and thereafter). Our goal is to reduce the relapse rate and move more patients to durable complete responses. We’ll have results later this year.

Stay tuned.

A Deep Dive Into The Use of Cytokines With CAR T Cells

Recent data from the 1H’2023 medical conferences: AACR, ASGCT, ASCO, EHA, Cellicon Valley

Author: Paul Rennert

Aleta Bio has invented, and develops, CAR T Engagers that work alongside cell therapeutics that treat cancers. As such we closely follow advances across the oncology therapeutic landscape, from CAR Ts to TCRs and TILs to antibody therapeutics, vaccine technologies, gene editing and of course basic T cell immunology. It is out of this last area that interest in cytokines as factors for CAR T cells (and in immuno-oncology generally) has emerged. I recent took a deep dive into the cytokine clinical space as part of our efforts to map how, and where, to deploy cytokines in the context of the CAR T Engager (CTE) platform. See the “Science” section for a primer on CTEs and the “Aleta-001” section for a description of our lead program, entering the clinic in Q3.

Use of cytokines with CAR-T cells or with other factors

The pre-clinical literature on the use of cytokines and other factors to improve CAR T cells is vast and has been extensively reviewed (1, 2). For the most part, I want to focus here is on recent clinical literature and presentations. The CAR T space incorporates cytokines in various ways, including during the manufacturing process. CARs that express cytokines or other factors after infusion into the patient are referred to as “armoured”. Beacon Intelligence recently profiled the armored-CAR landscape including CARs expressing cytokines:

Note that the dark blue bar that extends from the x-axis represent cytokines used with CAR-T cells.

These cytokines fall into a few categories:

a)     Cytokines that bind receptors that contain the common-gamma chain and signal through JAK/STAT pathways

b)    Pleotropic cytokines IL-12 and IL-18 and the pleotropic chemokine CCL5

c)     CCL19, 21: lymphoid structure chemokines

d)    Modifications to block the TGFb pathway

The chemokines and the TGFbeta pathway antagonists are not covered here; I’ll save those for another time. Several additional cytokines are mentioned in passing.

Cytokines that bind the common-gamma chain receptor

High systemic levels of the cytokines IL-2 and IL-15 demonstrate anti-tumor activity can induce unacceptable toxicity due to widespread expression of the IL-2 and IL-15 receptors; IL-7 is more muted and has biologic effects limited to T lymphocyte expansion and cell survival, and pro-inflammatory activity. There is a recent update on the use of these cytokines in cancer therapy (3).

Improving the utility of IL-2 has taken three distinct paths. One path is paved with IL-2 muteins engineered to bind selectively to subsets of the IL-2 receptors. This is a difficult strategy and has been slow to translate successfully despite sustained corporate effort (eg. Synthekine, Xilio, Werewolf, Nektar, etc). The second path involves fusions of IL-2 with antibody domains to various targets (tumor antigens, elements of the TME, costimulatory proteins), an interesting approach but one that requires overcoming technical, biological and clinical risks. The third is more biologically interesting and based on an understanding of IL-2 activities when presented with other factors.

Much of the cytokine literature is confusing in that both cell-expressed and exogenous cytokines are used. In some cases, these sources of cytokine matter, and this appears to be true of IL-2. A 2022 paper (4) demonstrated “that the capacity to manufacture IL-2 identifies constituents of the expanded CD8 T cell effector pool that display stem-like features, preferentially survive, rapidly attain memory traits, resist exhaustion, … cell-intrinsic synthesis of IL-2 by CD8 T cells attenuates the ability to receive IL-2–dependent STAT5 signals, thereby limiting terminal effector formation, endowing the IL-2–producing effector subset with superior protective powers.” In contrast, exogenous IL-2 drives signaling overwhelmingly through STAT5. The outcome in terms of T cell fate differ, and this may be why shorter CAR expansion ex vivo is emerging as a positive trend in CAR T manufacturing.

Activation-induced secretion of IL-2 by CAR T cells has been attempted to mimic the activity of normal activated T cells. Use of the NFAT-promoter and related strategies to trigger physiologic production of IL-2 have advanced preclinically (eg. (2)). In the meantime, the fact that most current CAR production utilizes extensive culture with exogenous IL-2 may limit the effectiveness of such controlled circuits.

Immunologic interactions suggest the utility of providing a source of second biologic signals that can work alongside IL-2 to optimally drive immune responses by triggering in vivo immune  cell interactions. As one example, CD40 ligand (CD40L) expression would normally be induced by TCR engagement which also triggers IL-2R CD25 upregulation and IL-2 expression. IL-2 signaling further upregulates CD40L expression. Thus, the IL-2 and CD40 pathways positively upregulate each other in a coordinated manner. Numerous papers describe the construction of CAR T cells that express or secrete CD40L primarily to “license” dendritic cells to upregulate adhesion molecules and present antigen. As one example, the Brentjen lab has described the effects of constitutive cell-surface expressed CD40L on CAR T cells in a syngeneic system (5). Further, CD40 activation via agonist antibody or viral CD40L expression has been well documented and clearly synergizes with IL-2 in preclinical models (eg. (6)). A CD40L-expressing CAR controlled by administration of a small molecule was licensed from Obsidian Therapeutics by Celegene/BMS (https://tinyurl.com/t9x9cnde). Less well understood, but of interest, is the immediate, albeit transient, expression of TNF by activated T cells. Both IL-2 and TNF are involved in activated T cell/dendritic cell cross-talk. This cross-talk is complicated by differences in CD4 and CD8 engagement, and the maturation/differentiation of DC subsets, and is under intense investigation.

Unmodified IL-2, TNF and IL-15 are toxic when given at effective levels systemically. Administration must be highly local, specifically, local to the CAR T cells, or expressed in an immune niche, or of modified forms of the cytokine. Several recent updates on use of novel forms of IL-2 illustrate this point. Ascendis Pharma presented a systemically administered “not-alpha” IL-2 +/- pembrolizumab at ASCO that ran into toxicity issues during Phase 1 dose escalation, with little sign of clinical activity (7). Cue Biopharma linked their not-alpha IL-2 to a soluble HLA module to direct IL-2 to HPV+ cancers. While toxicity was relatively mild, little clinical benefit was observed at the RP2D (8). Synthekine is developing human IL-2/IL-2Rβ-CAR orthogonal pairs whereby the mutant IL-2 can only bind the mutant receptor (see www.synthekine.com) as a way to further localize activity.

An example of IL-15 use in a CAR-T clinical setting was presented at ASGCT 2023. Here IL-15 was co-expressed with the CAR and toxicity was significant but not lethal. Some clinical responses were described, hinting that a therapeutic window was feasible. However, the safety switch was thrown to blunt toxicity, and this removes the CARs (9). To limit systemic distribution of fully active IL-15, membrane-bound forms are often used - typically these are complexes of IL-15. Precigen presented CAR-MUC16-mbIL-15 results in a Phase 1 study of platinum-resistant ovarian cancer and reported good tolerability and early efficacy signals (10). Many other examples have been presented in the pre-clinical literature and in the context of activating NK cells. Of note, membrane bound IL-15 signals through the IL-2b/yc receptor in trans, limiting the effects of IL-15 to cell/cell interactions. This interaction has been exploited to create soluble IL-15/IL-15Ra complexes that can be dosed as biologics, adding half-life extension, although so far this approach has had limited clinical success (11, 12). The advanced N-803 program from ImmunityBio using a so-called superagonist IL-15 biologic as part of a bladder cancer treatment regimen was recently rebuffed by the FDA, which declined the BLA application citing due to deficiencies observed during a pre-license inspection of the manufacturer’s third-party contract firms (see bit.ly/3nSoDnj). Other methods to limit systemic exposure include antibody fusions (PD-1, PD-L1, FAP, etc) and viral expression (eg. oncolytic and AAV).

In considering IL-15, the natural system consists of mbIL-15 cell surface complex, and short-lived secreted and non-complexed IL-15. IL-15 is generated by many cell types, including dendritic cells. Expression of IL-15 by the IL-15R+ T cells themselves is controversial, and the prevailing model favors trans presentation over cis presentation to T cells; the responding T population is predominately CD8+ T cells, which receive signals from IL-15 that support survival and memory cell production (13). Of note, the expression or appearance of IL-15 can be associated with tertiary lymphoid organ (TLO) development, a key feature of anti-tumor immunity (14–16). A particularly interesting finding is that copy number loss of the IL-15 (and other) genes is associated TLO status and outcome in ovarian cancer, a finding that supplies perhaps some rationale for the Precigen results (10).

The TLO finding is notable since it shows that IL-15 can be one of the signals that will not only promote CAR-T activity and expansion but that also support formation of an immune niche. This effect is mediated in part by Innate Lymphoid Cells (ILCs). ILCs were discovered by Reina Mebius when we worked together on lymph node genesis (17, 18). With this in mind one might explore the rationale for adding a local IL-15 source - eg. IL-15/IL-15Ra complex - to a local CD40L source, where these factors could be CAR-expressed or soluble. We may flesh this idea out a bit.

A recent write-up of the IL-2 and IL-15 competitive space can be found online (Evaluate Vantage). It’s clear these fields remain active despite the slow progress.

Efforts to use IL-7 therapeutically have expanded beyond the initial finding that this cytokine can safely be used to expand lymphocytes in patients to treat sepsis-induced lymphodepletion (19). Examples of IL-7 use in oncology include the long half-life biologic NT-17 (aka rhIL-7-hyFc; efineptakin-alfa) which has been administered + pembrolizumab in solid tumor clinical trials with early promising data (20) and has been administered alongside CAR T cells in preclinical models (21). In the context of use locally, it has been shown that while IL-7 can promote T cell development, expansion and survival, IL-15 may be required to ensure that T cell memory develops, and this may be true of CAR T cell also at least during ex-vivo culture (22). A very interesting recent study shows that “step-dosing” with IL-7 in primates can, in addition to causing peripheral lymphocyte proliferation, support DC/T cell maturation, induce CCL19, 20 and 22 expression, and promote immune interactions in lymph nodes (23). At ASH in December 2022, NeoImmuneTech presented early data on the use of a half-life extended form of IL-7 to boost CAR T cell numbers via an intramuscular injection 21 days post-CAR infusion. These data were interesting as the therapy was shown to safely increase CAR T number for several weeks (24). And to quickly circle back to IL-15, Nektar has a similar program for NKTR-255 IL-15 treatment given IV starting approximately 14 days post CAR T infusion (25).

In summary, IL-7 and IL-15 would appear to be interesting candidates for CAR expression or administration in soluble form. We should acknowledge that in any complex format - incorporating a cytokine with a targeting domain in a bispecific for example - two critical features must be examined: the first is the distribution of targets both in the tumor but also, critically, in lymphoid organs and other normal settings. The second is determining the optimal affinities for targets and getting the right balance for the in vivo setting where all binding events are subject to mass action effects. This may be an empirical exercise as it is difficult to model all known (and unknown) variables.

The pleotropic cytokines IL-12 and IL-18, and several other cytokines of interest

The IL-12 field is large and complex. The overarching goal here is to use engineering and/or drug delivery approaches that enhance IL-12 activity while reducing toxicity. These attempts are often thwarted by the unexpected finding that repetitive or chronic exposure to IL-12, even at tolerable dose levels, quickly reduces the cytokine’s impact on IFNgamma secretion, a key mechanism of IL-12 function.

As in the IL-2 field, the IL-12 field has taken multiple paths. Engineering IL-12 has been a common approach, perhaps best exemplified by work coming from the Garcia lab at Stanford (26), alongside developments in localized delivery. In that paper, protein engineering was used to selectively target IL-12 to T cells at the expense of NK cells. Updates on engineered IL-12 programs were presented at AACR and ASCO. AACR presentations included Synthekine, who licensed the Garcia/Stanford IP, and presented pre-clinical syngeneic mouse models to demonstrate the safety and efficacy of their IL-12 mutein (27). Note this is designed to avoid NK cell activation. Xilio has created a half-life extended and masked IL-12 that is freed for activity upon protease cleavage in the TME and also used pre-clinical syngeneic mouse models to show safety and efficacy vs wildtype IL-12 (28). Werewolf Therapeutics has a similar program (no recent data). Finally, Sonnet Therapeutics showed safety data from a Phase 1 trial of an anti-albumin scFv fusion with IL-12. In addition to extended PK, Sonnet claims that the presence of bound albumin will retain the protein in the TME due to interactions with GP60 and SPARC proteins (29). The drug appeared well tolerated but without monotherapy activity. On the other hand, a monovalent IL-12-Fc fusion was recently returned by Bristol Myers Squibb (BMS) to Dragonfly Therapeutics.

Localization of IL-12 to the TME directly, via gene therapy vectors or antibody delivery has been disappointing to data. AstraZeneca, citing safety/efficacy concerns, returned an IL-12mRNA/LNP formulation to Moderna; data presented at AACR suggest notable toxicity with limited efficacy when dosed intratumorally alongside systemic infusion of durvalumab (30). BNT131 and mRNA cocktail that produces soluble forms of IL-12, IL-15, IFN-α and GM-CSF was very recently discontinued in Phase 2 by BioNTech and Sanofi (https://tinyurl.com/yx9ymsfs). The MEDI1191 antibody, aka NHS-IL12, a fusion of IL-12 with a tumor-selective antibody, was abandoned by Merck KGgA. An IL-12 gene therapy approach from Oncosec, trialed with pembrolizumab, also reported poor results this year.

Other approaches in development with relevance for CAR-T are the numerous armored CAR concepts featuring cell surface retained IL-12 (26–29). Given the questionable safety/efficacy profile of numerous attempts to deliver IL-12 in the context of anti-tumor immunity I’m beginning to believe that this cytokine is too high risk to pursue. Nonetheless, CAR T technologies to consider are numerous:

-       IL-12 secreting MUC12-CAR T cells for ovarian cancer (35)

-       Local intra-tumoral delivery + CAR T added (36)

-       Inducible expression of IL-12 by CAR T cells, eg Obsidian Therapeutics

-       A comparison of tethering approaches (37)

IL-18 is a fascinating cytokine with a long history. It was pursued years ago by GSK, which abandoned the effort after serial failures. More recently, Aaron Ring of Yale, who did a post-graduate stint at aforementioned Garcia lab, showed that for secreted or soluble forms of IL-18 it was critical to mutate out binding to the soluble receptor decoy protein, IL-18bp, in order to maintain activity (38). The early data have been compelling, and the wider field is highly aware of the possibilities here (39–41). Carl June’s group at UPenn have published their preclinical work on the use of CAR T cells secreting a mutated IL-18 protein (42, 43). When the UPenn clinical data that were presented at the Cellicon Valley Conference in June are published, the intensity of interest is likely to increase. That program, CD19CAR-18, has been licensed by Novartis.

Other cytokines of interest include IL-21, FLT3L and GM-CSF. IL-21, like IL-2, 7 and 15, is variably used in CAR-T as a manufacturing factor, or as a CAR-T-secreted or tethered cytokine. The arguments for and limitations of its use are similar to those cytokines, ie. toxicity vs potency, with all the associated technologies to limit systemic exposure. FLT3L and GM-CSF activities are well known and well-understood. Indeed the Mooney lab at Harvard just published an interesting scaffold matrix that would deliver these factors in vivo (along with CPG) (44)

A few other things to consider.

Mutated IL-2 cytokines, or muteins, come with a variety of MOAs. I mentioned the “not-alpha” IL-2 program from Ascendis Pharma, the goal here is to avoid triggering IL-2 receptor complexes that express the alpha receptor chain, CD25. The proposed MOA is to preferentially expand effector T cells while not expanding regulatory T cells. As noted above, toxicity without efficacy was seen in the Ascendis clinical program. Other not-alpha programs include Nektar Therapeutics PEGylated version of IL-2 (failed in multiple clinical trials), Xilio’s masked IL-2 mutein, and programs from Sanofi, Merck, BioNTech and many others (45). In contrast, Synthekine seeks to selectively target the alpha/beta form of the IL-2 receptor to activate T cells selectively, avoiding NK cells. None of these approaches have yielded positive clinical data to date.

From the perspective of establishing baseline immune stimulation, CD40 agonists appear well suited. Like other immune agonists that have clear toxicity issues like 4-1BB (46), CD40 agonist toxicity is both on-target (eg. mainly CRS, hyper-immunity) and off-target (hepatotoxicity) suggesting locoregional delivery will be optimal (47). Other agonists in this space have shown minimal activity: OX40 (48) and CD27 (49).

CD40 activity has been associated with efficacy in early clinical studies. Multiple trials are ongoing and recent results include:

-       Clear responses reported by SeaGen, some durable, in pancreatic cancer (combination setting with pembro/chemo) (https://www.cancernetwork.com/view/sea-cd40-chemo-combo-yields-anti-tumor-efficacy-in-pdac)

-       MOA data in PDAC from Apexigen (apexigen link)

-       Early clinical responses in SCCHN, in combination with pembrolizumab (50)

CD40 agonism as monotherapy has had modest effect, so pairing with a T cell stimulatory pathway makes sense (ie. to essentially achieve an effect similar to PD-(L)-1 pathway antagonism). Since signaling through STAT5 can overcome PD-1 mediated immune suppression, one or more of the IL-2, IL-7, IL-15 group could be incorporated. One might for example express CD40L on the CAR and then also add a second cytokine to the CAR or as a soluble factor. Beyond IL-2, IL-7 and IL-15, I believe IL-12 needs further derisking in the clinic as noted earlier. This is also the case with IL-21, as there is just less data (see the Werewolf program for an example (51). Other pathways of interest include FLT3L, GM-CSF and IFNalpha (Werewolf, (52).

In combination with CAR T cells, interaction with APCs that could present the relevant antigen and/or provide costimulatory signals appear desirable, and engaging endogenous T cells that might further drive anti-tumor immunity to enhance CAR T activity would also be desirable. The use of IL-15 to drive memory formation is a reasonable T cell centric idea, perhaps with FLT3L to attract monocytes/DCs or anti-CD40 or CD40L to engage B cells and DCs. The T cell expansion literature is replete with T cell cytokine combinations (2+7, 2+15, 2+21, 15+12, the list goes on) but I think the emphasis on locoregional activation could help focus on more immune niche-supporting factors, and here IL-15 and either FLT3L or agonist CD40 stand out to me. The other potential class here is chemokines, and these bring the complexity of needing to establish a stable concentration gradient. I don’t know the technology needed how to do that engineering but I’ll bet the nanoparticle and artificial scaffold engineers have an idea or two.

Stay tuned.

References

1. Hawkins ER, D’Souza RR, Klampatsa A. Armored CAR T-Cells: The Next Chapter in T-Cell Cancer Immunotherapy. Biologics 2021;15:95–105.

2. Harrison AJ, Du X, von Scheidt B, Kershaw MH, Slaney CY. Enhancing co-stimulation of CAR T cells to improve treatment outcomes in solid cancers. Immunother Adv 2021;1(1):ltab016.

3. Wolfarth AA et al. Advancements of Common Gamma-Chain Family Cytokines in Cancer Immunotherapy [Internet]. Immune Network 2022;22(1). doi:10.4110/in.2022.22.e5

4. Kahan SM et al. Intrinsic IL-2 production by effector CD8 T cells affects IL-2 signaling and promotes fate decisions, stemness, and protection. Science Immunology 2022;7(68):eabl6322.

5. Kuhn NF et al. CD40 Ligand-Modified Chimeric Antigen Receptor T Cells Enhance Antitumor Function by Eliciting an Endogenous Antitumor Response. Cancer Cell 2019;35(3):473-488.e6.

6. Zhang Y et al. Chimeric antigen receptor T cells engineered to secrete CD40 agonist antibodies enhance antitumor efficacy. Journal of Translational Medicine 2021;19(1):82.

7. Starodub A et al. Phase 1/2 dose escalation and dose expansion study of TransCon IL-2 β/γ alone or in combination with pembrolizumab: Initial results from dose escalation.. JCO 2023;41(16_suppl):e14597–e14597.

8. Chung CH et al. A phase 1 dose-escalation and expansion study of CUE-101, a novel HPV16 E7-pHLA-IL2-Fc fusion protein, given as monotherapy and in combination with pembrolizumab in patients with recurrent/metastatic HPV16+ head and neck cancer.. JCO 2023;41(16_suppl):6013–6013.

9. Abstract Details | ASGCT Annual Meeting [Internet]https://annualmeeting.asgct.org/abstracts/abstract-details?abstractId=15009. cited July 2, 2023

10. Liao JB et al. Phase 1/1b study of PRGN-3005 autologous UltraCAR-T cells manufactured overnight for infusion next day to advanced stage platinum resistant ovarian cancer patients.. JCO 2023;41(16_suppl):5590–5590.

11. Chamie K et al. Final clinical results of pivotal trial of IL-15RαFc superagonist N-803 with BCG in BCG-unresponsive CIS and papillary nonmuscle-invasive bladder cancer (NMIBC).. JCO 2022;40(16_suppl):4508–4508.

12. Knudson KM, Hodge JW, Schlom J, Gameiro SR. Rationale for IL-15 superagonists in cancer immunotherapy. Expert Opinion on Biological Therapy 2020;20(7):705–709.

13. Park J-Y, Ligons DL, Park J-H. Out-sourcing for Trans-presentation: Assessing T Cell Intrinsic and Extrinsic IL-15 Expression with Il15 Gene Reporter Mice [Internet]. Immune Network 2018;18(1). doi:10.4110/in.2018.18.e13

14. Aoyama S, Nakagawa R, Mulé JJ, Mailloux AW. Inducible Tertiary Lymphoid Structures: Promise and Challenges for Translating a New Class of Immunotherapy [Internet]. Frontiers in Immunology 2021;12.https://www.frontiersin.org/articles/10.3389/fimmu.2021.675538. cited July 2, 2023

15. He T et al. Oncolytic adenovirus promotes vascular normalization and nonclassical tertiary lymphoid structure formation through STING-mediated DC activation. Oncoimmunology 11(1):2093054.

16. Sautès-Fridman C et al. Tertiary Lymphoid Structures in Cancers: Prognostic Value, Regulation, and Manipulation for Therapeutic Intervention. Front Immunol 2016;7:407.

17. Mebius RE, Rennert P, Weissman IL. Developing lymph nodes collect CD4+CD3- LTbeta+ cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells. Immunity 1997;7(4):493–504.

18. Bar-Ephraïm YE, Mebius RE. Innate lymphoid cells in secondary lymphoid organs. Immunol Rev 2016;271(1):185–199.

19. Daix T et al. Intravenously administered interleukin-7 to reverse lymphopenia in patients with septic shock: a double-blind, randomized, placebo-controlled trial. Annals of Intensive Care 2023;13(1):17.

20. Naing A et al. Efficacy and safety of NT-I7, long-acting interleukin-7, plus pembrolizumab in patients with advanced solid tumors: Results from the phase 2a study.. JCO 2022;40(16_suppl):2514–2514.

21. Kim MY et al. A long-acting interleukin-7, rhIL-7-hyFc, enhances CAR T cell expansion, persistence, and anti-tumor activity. Nat Commun 2022;13(1):3296.

22. Zhou J et al. Chimeric antigen receptor T (CAR-T) cells expanded with IL-7/IL-15 mediate superior antitumor effects. Protein Cell 2019;10(10):764–769.

23. Pandit H et al. Step-dose IL-7 treatment promotes systemic expansion of T cells and alters immune cell landscape in blood and lymph nodes [Internet]. iScience 2023;26(2). doi:10.1016/j.isci.2023.105929

24. Ghobadi A et al. A Phase 1b Dose Expansion Study Evaluating Safety, Preliminary Anti-Tumor Activity, and Accelerated T Cell Reconstitution with NT-I7 (Efineptakin Alfa), a Long-Acting Human IL-7, Administered Following Tisagenlecleucel in Subjects with Relapsed/Refractory Large B-Cell Lymphoma. Blood 2022;140(Supplement 1):10366–10367.

25. Perales M-A et al. A Phase 2/3, Randomized, Double Blind, Placebo-Controlled, Multicenter Study of NKTR-255 Vs Placebo Following CD-19 Directed CAR-T Therapy in Patients with Relapsed/Refractory Large B-Cell Lymphoma. Blood 2022;140(Supplement 1):7488–7490.

26. Cr G et al. Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells [Internet]. Cell 2021;184(4). doi:10.1016/j.cell.2021.01.018

27. Koliesnik I et al. Abstract 1833: Novel IL-12 Partial Agonist For Cancer Immunotherapy Avoids NK-cell Mediated Toxicity. Cancer Research 2023;83(7_Supplement):1833.

28. Malkova N et al. Abstract 587: A half-life extended, tumor-activated IL-12 increased the infiltration of effector immune cells into the tumor microenvironment and demonstrated anti-tumor activity in syngeneic mouse models. Cancer Research 2023;83(7_Supplement):587.

29. Chawla SP et al. Abstract CT245: Clinical development of a novel form of interleukin-12 with extended pharmacokinetics. Cancer Research 2023;83(8_Supplement):CT245.

30. Intratumoral (IT) MEDI1191 + durvalumab (D): Update on the first-in-human study in advanced solid tumors [Internet]https://www.abstractsonline.com/pp8/#!/10828/presentation/10246. cited July 4, 2023

31. Jones DS et al. Cell surface–tethered IL-12 repolarizes the tumor immune microenvironment to enhance the efficacy of adoptive T cell therapy. Science Advances 2022;8(17):eabi8075.

32. Zhang L et al. Enhanced efficacy and limited systemic cytokine exposure with membrane-anchored interleukin-12 T-cell therapy in murine tumor models. J Immunother Cancer 2020;8(1):e000210.

33. Luo Y et al. IL-12 nanochaperone-engineered CAR T cell for robust tumor-immunotherapy. Biomaterials 2022;281:121341.

34. Holder PG et al. Engineering interferons and interleukins for cancer immunotherapy. Advanced Drug Delivery Reviews 2022;182:114112.

35. Koneru M, O’Cearbhaill R, Pendharkar S, Spriggs DR, Brentjens RJ. A phase I clinical trial of adoptive T cell therapy using IL-12 secreting MUC-16ecto directed chimeric antigen receptors for recurrent ovarian cancer. Journal of Translational Medicine 2015;13(1):102.

36. Agliardi G et al. Intratumoral IL-12 delivery empowers CAR-T cell immunotherapy in a pre-clinical model of glioblastoma. Nat Commun 2021;12(1):444.

37. Hu J et al. Cell membrane-anchored and tumor-targeted IL-12 (attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors. J Immunother Cancer 2022;10(1):e003633.

38. Zhou T et al. IL-18BP is a secreted immune checkpoint and barrier to IL-18 immunotherapy. Nature 2020;583(7817):609–614.

39. Jaspers JE et al. IL-18-secreting CAR T cells targeting DLL3 are highly effective in small cell lung cancer models. J Clin Invest 2023;133(9):e166028.

40. Glienke W et al. GMP-Compliant Manufacturing of TRUCKs: CAR T Cells targeting GD2 and Releasing Inducible IL-18. Front Immunol 2022;13:839783.

41. Olivera I et al. mRNAs encoding IL-12 and a decoy-resistant variant of IL-18 synergize to engineer T cells for efficacious intratumoral adoptive immunotherapy. Cell Rep Med 2023;4(3):100978.

42. Carroll RG et al. Distinct effects of IL-18 on the engraftment and function of human effector CD8 T cells and regulatory T cells. PLoS One 2008;3(9):e3289.

43. Hu B et al. Augmentation of Antitumor Immunity by Human and Mouse CAR T Cells Secreting IL-18. Cell Rep 2017;20(13):3025–3033.

44. Adu-Berchie K et al. Adoptive T cell transfer and host antigen-presenting cell recruitment with cryogel scaffolds promotes long-term protection against solid tumors. Nat Commun 2023;14(1):3546.

45. Dolgin E. IL-2 upgrades show promise at ASCO. Nature Biotechnology 2022;40(7):986–988.

46. Su TT, Gao X, Wang J. A Tumor-Localized Approach to Bypass Anti-4-1BB Immuno-Toxicity. Clinical Cancer Research 2019;25(19):5732–5734.

47. Salomon R, Dahan R. Next Generation CD40 Agonistic Antibodies for Cancer Immunotherapy. Front Immunol 2022;13:940674.

48. Gutierrez M et al. OX40 Agonist BMS-986178 Alone or in Combination With Nivolumab and/or Ipilimumab in Patients With Advanced Solid Tumors. Clinical Cancer Research 2021;27(2):460–472.

49. Sanborn RE et al. Safety, tolerability and efficacy of agonist anti-CD27 antibody (varlilumab) administered in combination with anti-PD-1 (nivolumab) in advanced solid tumors. J Immunother Cancer 2022;10(8):e005147.

50. Sanborn R et al. 596 Results from a phase 1 study of CDX-1140, a fully human anti-CD40 agonist monoclonal antibody (mAb), in combination with pembrolizumab [Internet]. J Immunother Cancer 2022;10(Suppl 2). doi:10.1136/jitc-2022-SITC2022.0596

51. Sullivan JM et al. Abstract 1829: Generation of IL-21 INDUKINETM molecules for the treatment of cancer. Cancer Research 2023;83(7_Supplement):1829.

52. Nirschl CJ et al. Abstract 1817: WTX-613, (JZP898) a selectively activated IFNα INDUKINETM molecule, reprograms the tumor microenvironment and generates robust anti-tumor immunity as a monotherapy and in combination with checkpoint inhibitors. Cancer Research 2023;83(7_Supplement):1817.