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.