Granzyme B PET Imaging of Immune Checkpoint Inhibitor Combinations in Colon Cancer Phenotypes

Immune checkpoint receptors are crucial molecules that regulate the immune system, dampening T cell activation. Tumours exploit these checkpoints to evade the immune system. Immune checkpoint inhibitors (ICIs) have changed the landscape of cancer treatment; however, responses vary depending on tumour type and only a minority of patients show a durable response [1‐4]. Combination ICI therapy is being actively pursued as a strategy to improve the rate of response. PD-1 and CTLA-4, the two most commonly studied checkpoints, are not functionally redundant and while they both act to modulate distinct populations of tumour-infiltrating lymphocytes (TILs), including CD4+, CD8+, regulatory T (T_reg) cells, they do so with different mechanistic profiles. Thus, combination therapies may act in a complementary or synergistic fashion depending on the tumour microenvironment [5]. However, the biology surrounding combination therapies is not well understood and combined use may also increase the incidence of potentially severe side effects. Recent studies using a combined PD-1/CTLA-4 strategy have shown increased immune-related side effects and unexpected off target effects including cardiac toxicity [6]. It is hoped that newer ICIs may improve efficacy when combined with established inhibitors such as PD-1, while reducing unwanted side effects. The negative immune checkpoints TIM-3 (T cell immunoglobulin and mucin domain-3) and LAG-3 (lymphocyte-associated gene 3) are co-expressed with PD-1 on T cells and are being actively assessed in the clinic [7‐9]. TIM-3 is expressed on numerous immune cell types including T cells, dendritic cells, and macrophages and is thought to be involved in immune tolerance by mediating T cell exhaustion [8]. LAG-3 is also a checkpoint modulator, is also thought to be involved in immune tolerance by mediating T cell exhaustion [7] and has been shown to be expressed on expressed on natural killer (NK), activated T cells and T_reg TILs [5, 10]. Alternative strategies include targeting OX40, a T cell co-stimulator expressed on CD4 and CD8 T cells during antigen specific priming which leads to T cell expansion and differentiation. OX40 antibody agonists have shown significant therapeutic efficacy both as monotherapies and in combination with PD-1 blockade leading to significant tumour regression [11]. Certain phenotypes, such as microsatellite instability (MSI), mutational burden, and neoantigenic load have been demonstrated to enrich response to PD-1 monotherapy [2, 12]. Currently, there is little evidence to determine whether these will also enrich response to the newer immune therapies and importantly no way to accurately predict individual patient response. Accurate stratification of treatment response to monotherapies and especially combination therapies, across all tumour phenotypes, is paramount to ensure safe and effective patient management.

Currently, there is a dearth of biomarkers capable of accurately stratifying ICI treatment response. Non-invasive imaging of the tumour microenvironment is being intensively researched to accurately assess immune therapies. Several studies have shown that TILs are necessary for checkpoint inhibitor efficacy; thus, radiopharmaceuticals have been developed to target T lymphocyte populations including CD3 and CD8. The presence of TILs alone, however, may not be enough to accurately stratify response due to immune cell regulation or tolerance [13‐15]. Granzyme B is released upon activation of cytotoxic CD8+ T cells, providing information about T cell location and tumouricidal activity. A ⁶⁸Ga-labelled peptide that targets GZB has been shown to reliably stratify response to ICIs in preclinical tumour models [16‐18]. In the current study, we have developed and assessed a new fluorine-labelled derivative, [¹⁸F]AlF-mNOTA-GZP, to improve PET imaging sensitivity and spatial resolution [19]. The ability of [¹⁸F]AlF-mNOTA-GZP to stratify immunotherapy response was assessed in response to ICI monotherapy and combined ICI therapies in CT26 and MC38 syngeneic colon cancer models, frequently associated with MSI-low phenotype, and MSI-high phenotype [20], respectively. PET imaging results were then correlated to differences in TIL subpopulations by flow cytometry.

Immune checkpoint inhibitor (ICI) monotherapy has been shown to trigger activation of compensatory T cell-associated checkpoints [5], such as PD-1 blockade upregulating TIM-3, an effect that could perhaps be mitigated by combined therapy. Emerging evidence supports the use of combined ICI regimens, although toxicity profiles, especially those associated with combined CTLA-4 blockade, may be of concern [3]. With the substantial number of clinical trials evaluating ICI combination therapies, the ability to rapidly and reliably assess therapy efficacy is paramount to ensure safe patient management.

Non-invasive molecular imaging of immunotherapy has predominantly focused on the expression of immunologically relevant targets within the tumour microenvironment. The majority of studies have investigated the utility of measuring PD-L1 expression to predict therapy response and have shown that while they may have excellent utility for patient selection, PD-L1 expression is dynamic and context dependent and high PD-L1 expression does not guarantee therapy response [23, 24]. Immunotherapy success is dependent on TILs and numerous biomarkers have been developed to assess changes in specific T cell subsets by the specific targeting of cell-surface CD molecule expression (CD3, CD8) [13‐15]; however, high TIL expression does not guarantee therapy response either, due to tolerance and regulatory mechanisms [25, 26]. Detection of molecules that represent an effector T cell phenotype has proven to be more successful. The radiolabelled GZB targeting peptide [⁶⁸Ga]Ga-mNOTA-GZP has been shown to be well correlated with immunotherapy efficacy [16‐18]. In the current study, we have radiolabelled the GZB targeting peptide using [¹⁸F]AlF to improve PET imaging duration of use, sensitivity and spatial resolution [19]. The addition of [¹⁸F]AlF did not substantially change the enzymatic activity of the peptide (Supplemental Fig. S1), nor did it change the in vivo uptake and excretion kinetics compared to the ⁶⁸Ga-labelled peptide (Supplemental Fig. S2a and b). Furthermore, when compared to our previously published data on the ⁶⁸Ga-labelled peptide, very similar tracer uptake was observed in CT26 tumours in both ICI treatment responder and non-responder groups [18].

Interestingly, both radiopharmaceutical uptake and response to therapy was very different in the CT26 and MC38 tumours. These differences are likely due to the MSI phenotype found in MC38 tumours. MSI or deficiency of DNA mismatch repair system (MMR) occurs in approximately 15 % of colorectal cancers [20] and renders cancer cells unable to correct errors that occur at microsatellite regions during DNA replication. This leads to the accumulation of mutations which likely increase the amount and immunogenicity of neoantigens [12, 27‐29]. Even with such an immunostimulatory environment, only ~ 50 % of MSI colorectal cancers respond to classical ICIs clinically while MSI negative colorectal cancers generally do not respond [20]. The effects of this immunostimulatory environment can be clearly observed in the tumour responses to ICIs and subsequent [¹⁸F]AlF-mNOTA-GZP uptake. Overall, the CT26 tumours showed a modest (~ 40 %) response to ⍺PD1 monotherapy and an improved (~ 70 %) response to ⍺CTLA4 or ⍺OX40 monotherapy or combined ⍺PD1 with ⍺CTLA4 or ⍺PD1 with ⍺OX40 therapy, as determined by tumour growth inhibition. The newer combination therapies ⍺TIM3 and ⍺LAG3, however, did not effectively inhibit tumour growth in the CT26 tumour bearing mice when used as monotherapies. Furthermore, neither ⍺TIM3 or ⍺LAG3, effectively inhibited tumour growth when used in combination with ⍺PD1, despite ⍺PD1 monotherapy proving efficacious in 40 % of tumours when used as a monotherapy. This lack of response may be due to the relatively low total number of CT26 resident cytotoxic T cells, despite tumour-infiltrating CD8+ cells having been shown to express TIM-3 and LAG-3 [19, 30]. The low number of T cells associated with the CT26 tumours leads to a relatively low tumour uptake of [¹⁸F]AlF-mNOTA-GZP at ~ 0.2%ID/g in non-responsive tumours, peaking at ~ 0.6%ID/g in treatment responsive tumours. The MC38 tumours, with a much greater number of cytotoxic T cells, displayed a higher response rate to all therapies (~ 70–90 % response to ⍺PD1, ⍺CTLA4, ⍺OX40 or combinations and successfully responded to ⍺TIM3, ⍺LAG3 monotherapies or combinations (~ 40–60 %). [¹⁸F]AlF-mNOTA-GZP uptake was similarly affected by the immunostimulatory environment, with significantly higher uptake in all the MC38 tumours (in controls, TRs and TNRs alike) ranging from ~ 0.6%ID/g in non-responsive tumours (levels comparable to peak responders in the CT26 tumour type) and up to ~ 1.1%ID/g in treatment-responsive tumours. In both tumour types, uptake of [¹⁸F]AlF-mNOTA-GZP in TRs were positively correlated with CD3+, CD8+ and CD8+GZB+ TILs (Figs. 4 and 5), however, the variable levels of immune cells prevalent in the MSI high MC38 tumours, obscured the changes in T cell populations diminishing the correlation between TILs and [¹⁸F]AlF-mNOTA-GZP uptake when compared to the MSI-low CT26 tumours. The difference in tumour immunostimulatory environments may be a limiting factor affecting the utility of [¹⁸F]AlF-mNOTA-GZP, potentially obfuscating response stratification in immune-sensitive tumours.

Furthermore, while the number of non-responsive MC38 tumours was relatively low, those that did not respond displayed an intriguing reduction in CD8+ TILs compared with the control treated group, potentially linked with the regulatory suppression mechanisms that caused these tumours to be non-responsive. While a small increase in CD4+ FOXP3+ CD25+ Tregs was observed in the MC38 TNRs when compared to the control treated tumours this was not significant. Certainly, the presence of mutations can influence the function of several cellular processes and these may include those that are designed to nullify the host response.

In summary, the current study shows that in vivo molecular imaging of intra-tumoural granzyme B using [¹⁸F]AlF-mNOTA-GZP is able to stratify tumour response to a range of ICIs administered to preclinical colon cancer models given as monotherapies or in combination. However, care should be taken in interpreting the images, as tracer uptake can be significantly affected by pre-existing phenotypic abnormalities. Further studies will be needed to elucidate other factors affecting the interpretation of granzyme B targeting tracers before they could be used for stratification of patient response across multiple tumour phenotypes.