Image-Guided Interventional Radiological Delivery of Chimeric Antigen Receptor (CAR) T Cells for Pleural Malignancies in a Phase I/II Clinical Trial

Abstract

Objectives:

We describe techniques and results of image-guided delivery of mesothelin-targeted chimeric antigen receptor (CAR) T cells in patients with pleural malignancies in a phase I/II trial (ClinicalTrials.gov: NCT02414269).

Materials and Methods:

Patients without a pleural catheter or who lack effusion for insertion of a catheter (31 of 41) were administered intrapleural CAR T cells by interventional radiologists under image guidance by computed tomography or ultrasound. CAR T cells were administered through a needle in an accessible pleural loculation (intracavitary) or following an induced loculated artificial pneumothorax. In patients where intracavitary infusion was not feasible, CAR T cells were injected via percutaneous approach either surrounding and/or in the pleural nodule/thickening (intratumoral). Pre- and post-procedural clinical, laboratory, and imaging findings were assessed.

Results:

CAR T cells were administered intrapleurally in 31 patients (33 procedures, 2 patients were administered a second dose) with successful delivery of planned dose (10–186 mL); 14/33 (42%) intracavitary and 19/33 (58%) intratumoral. All procedures were completed within 2 hours of T-cell thawing. There were no procedure-related adverse events greater than grade 1 (1 in 3 patients had prior ipsilateral pleural fusion procedures). The most common imaging finding was ground glass opacities with interlobular septal thickening and/or consolidation, observed in 12/33 (36%) procedures. There was no difference in the incidence of fever, CRP, IL-6, and peak vector copy number in the peripheral blood between infusion methods.

Conclusion:

Image-guided intrapleural delivery of CAR T cells using intracavitary or intratumoral routes is feasible, repeatable and safe across anatomically variable pleural cancers.

1. INTRODUCTION¹

Cancer therapeutics including chemotherapy and immune checkpoint inhibitors are administered systemically by intravenous infusion allowing for the distribution of the administered drug through the circulation. Adoptive cell therapy by chimeric antigen receptor (CAR) T cells demonstrated impressive results with intravenous infusion of CD19 and B-cell maturation antigen—cancer-cell surface antigen targeted CAR T cells for hematological malignancies [1–3]. However, we and others have shown the limitations of intravenous administration of CAR T cells to treat solid tumors; known barriers include pulmonary sequestration of adoptively transferred cells that are activated by cytokine beads during manufacturing with ensuing pulmonary inflammation, potential “on-target off-tumor” toxicity within the lung, limited CAR T-cell trafficking, and infiltration into the tumor compounded by unique microenvironment of the tumor embedded organ [4–8]. Biological therapies such as oncolytic viruses have been delivered intratumoral to promote their infiltration into the tumor, to prevent neutralization by preexisting antibodies in the systemic circulation, and to augment their efficacy [9–11], and raised interest in this delivery approach with cancer immunotherapies [12–15].

Case reports demonstrated the feasibility of regional delivery of CAR T cells via intracranial, intrahepatic, or intraperitoneal routes [16–19]. While these approaches have used catheter-directed delivery of cells, image-guided procedures performed by interventional radiologists allow for the reach of a wide range of organs, including the liver [20], lung [21], pleura [22], and peritoneum [23], with high precision, facilitating intratumoral immunotherapies for solid organ or diffuse cavitary embedded tumors [24]. However, to date, there is no description of the techniques and results for regional delivery of immunotherapy including CAR T cells in a large series of patients; several approaches are under investigation [24–28].

Pleural cancers resulting from primary malignant pleural mesothelioma (MPM) or metastatic lung and breast cancers affect >150,000 patients a year within the U.S. alone and are associated with poor clinical outcomes [29–31]. MPM’s unique characteristic of locoregional invasion contributing to the aggressiveness with lower incidence of distant metastases provides an opportunity to deliver gene-engineered immune cells directly to the pleural cavity [32,33]. In addition to the clinical rationale to deliver CAR T cells locoregionally, our group has shown the immunological benefits from immediate antigen activation and proliferation of CAR T cells as well as augmented CD4 helper function following intrapleural administration of mesothelin, a cancer-cell surface antigen-targeted CAR T cells in an orthotopic MPM mouse model [4,19]. Intrapleural administration demonstrated superior anti-tumor efficacy even at a 30-fold lower dose as well as systemic immunity compared to intravenous administration [4]. Our recent phase I trial confirmed that regionally delivered mesothelin-targeted CAR T cells were able to treat the pleural tumor and persist in systemic circulation [19]. Patients with pleural malignancies can have variable clinical presentation, including pleural masses, heterogenous pleural thickening (parietal, visceral or both), pleural nodules in the fissure, pleural effusion (generalized or loculated), or a combination of these, complicating the feasibility of regional intervention and delivery of cells, especially among patients previously treated with surgery or radiotherapy to the pleura [34].

Given the significance of a personalized approach / technique in regional delivery of immunotherapy to ensure successful administration of the intended dose and homogeneity of treatment delivery in clinical trials [26,35], the primary aim of this study is to describe the successful techniques of image-guided, interventional radiological (IR) delivery of mesothelin-targeted CAR T cells in patients with pleural malignancies in our phase I/II clinical trial (ClinicalTrials.gov: NCT02414269), with a focus on administration routes, target selection criteria, post-procedural imaging findings, and safety. We demonstrate that regional intrathoracic delivery of a large volume of therapeutic agent is feasible, safe and effective in patients with pleural malignancies. We further propose an algorithmic approach to deliver agents to the pleural cavity using IR expertise.

2. MATERIALS and METHODS

2.1. Study Design

This is an open-label, dose-escalating, single-center, phase I/II study of mesothelin-targeted CAR T cells in patients with previously treated histologically proven pleural cancer from MPM, metastatic lung cancer, or metastatic breast cancer (Trial registration number: NCT02414269, study protocol provided in the Supplementary Material). Patients with mesothelin expression of at least 10% of tumor cells on immunohistochemical analysis and/or patients with epithelioid mesothelioma with serum soluble mesothelin-related peptides > 1.0 nm/L were eligible. Following a single dose of preconditioning cyclophosphamide (1500 mg/m²), mesothelin-targeted CAR T cells (0.3M-60M CAR T cells/kg) with the iCaspase-9 safety gene were administered intrapleurally either through a pleural catheter or via IR-guided imaging. Among 41 patients treated to date, this report is a subgroup analysis of 31 patients (33 procedures, 2 patients received repeat administration) who underwent image-guided infusion of CAR T cells performed by IR.

2.2. Study Oversight

The study protocol and amendments were approved by the institutional review board at Memorial Sloan Kettering Cancer Center. All patients provided written informed consent. Response and toxicity outcomes were reviewed by an independent committee established by the institutional Clinical Research Oversight Committee to manage potential conflicts of interest in the interpretation of responses. No one who is not an author contributed to the writing of the manuscript.

2.3. Image-guided Infusion of CAR T Cells

Prior to the procedure, contraindications for percutaneous solid organ biopsies were ruled out: severe coagulopathy that cannot be corrected, namely platelet levels < 50K/mcL or international normalized ratio > 1.5, or hemodynamic instability [36]. All patients who do not have an indwelling pleural catheter or lack generalized pleural effusion for insertion of pleural catheter (31/41) were referred to the IR clinic to assess the feasibility and to obtain informed consent.

CAR T cells were infused in the pleural space (intracavitary infusion) and/or directly surrounding/into the tumor (intratumoral infusion) through a needle or a catheter placed under imaging guidance by interventional radiologists with at least 5 years of experience in performing intrathoracic interventional procedures, including lung and pleural biopsies and thermal ablations. Procedures were performed under moderate intravenous sedation, local anesthesia, and continuous non-invasive monitoring of vital signs. Patients were positioned supine if the percutaneous access was anterior or lateral and in prone position otherwise.

2.4. Intratumoral and Intracavitary Infusion Routes

Chest computed tomography (CT) or fluorine-18-fluorodeoxyglucose positron emission tomography (PET) CT scan performed within one month of the procedure were reviewed by the interventional radiologist and principal investigator of the trial to select the appropriate target. Following confirmation that there were no issues following cyclophosphamide lymphodepletion that preclude IR procedure, the timing of IR procedure was coordinated with IR staff, principal investigator and cellular therapeutics center nursing staff as manufactured CAR T cells have to be infused within 3 hours of thawing and cannot be frozen back (protocol in Supplementary Material). Choice of appropriate infusion route was determined based on tumoral presentation (different tumoral presentations are presented in Fig. 1A–D), tumor size, and uptake on PET CT.

Figure 1. Various clinical presentations and image-guided intracavitary and intratumoral infusion routes of CAR T cells for pleural malignancies.

(A) Contrast enhanced chest CT showing diffuse right pleural thickening, with multiple nodules of various sizes (arrowheads). Note the extension of the pleural thickening to the interlobar fissure (long arrow). (B) Contrast enhanced chest CT showing a single right pleural mass (arrowhead). (C) This patient presented with only a small right pleural effusion (arrowhead). (D) The most challenging disease presentations were patients with only minimal pleural thickening (arrowhead), here measured at 9 mm thickness. (E) CAR T cells were injected into the pleural space (arrowhead), as shown in this case (Patient 37) where a 5Fr Yueh needle was inserted (arrow) under ultrasound guidance. (F) Axial non-contrast chest CT scan (Patient 39) after a successful pleural expansion showing the artificially induced pneumothorax (arrowhead) and a catheter placed within the newly expanded pleural cavity (arrow). (G) Axial non contrast chest CT scan (Patient 13) showing direct intratumoral infusion with a 17G needle (arrow) inserted percutaneously from an anterior intercostal approach directly into a right pleural mass (arrowhead) that was extending to the fissure. (H) Axial non contrast chest CT scan (Patient 19) showing another intratumoral infusion target, here a 12mm pleural thickening (arrowhead), also targeted with a 17G needle (arrow) using a posterior approach.

For intracavitary infusion, if the patient presented with a pleural effusion (free or loculated), a needle or a catheter was placed under image guidance in the pleural space to infuse CAR T cells (Fig. 1E). Prior to the administration of CAR T cells, pleural fluid was completely drained. When pleural effusion was absent or estimated too small by the operator, a pleural expansion could be attempted in an aim to provide enough space for the volume of the solution to be injected. Pleural expansion consisted of insufflating air in the pleural space through a percutaneously inserted needle. Presence of a small pneumothorax confirmed success and, consequently, the operator placed a pleural catheter into this induced pleural space prior to infusion (Fig. 1F).

For intratumoral infusion, if intracavitary infusion could not be performed, CAR T cells were directly injected via percutaneous access either surrounding and/or in the pleural nodule/mass (Fig. 1G) or in the pleural thickening (Fig. 1H). In case of multiple nodules, those with the highest metabolic activity on PET CT and largest diameter accessible for infusion were selected. Concerning injections into pleural thickening, the needle was inserted along the pleural plane as previously described for pleural biopsies to ensure a successful delivery of CAR T cells even when the pleural thickening was minimal [22].

Choice of the appropriate imaging guidance and material were at the discretion of the operator, but the desired final in-target position was verified by CT imaging in all procedures [37,38]. Once the position of the final needle/catheter was confirmed on CT, CAR T cells were consequently injected by the study principal investigator (P.S.A.) or by the intervention radiologist. All patients had a post-infusion CT and chest radiography immediately and 2 hours after the CAR T-cell infusion to rule out any expanding pneumothorax or other procedural complications.

2.5. Technical Success

Technical success was defined as the ability to deliver complete volume of CAR T cells (10–186 mL; increasing volume with increasing T-cell dose by cohort) to the desired target. Patients medical records were reviewed for the confounding variables that could influence the technical success: previous treatments (surgery, radiotherapy, and/or regional infusion of CAR T cells), tumoral presentation on chest CT, infusion route, imaging guidance modality, material used to inject CAR T cells, volume and dose infused, anesthesia time, as well as intra- and post-procedural imaging findings.

2.6. Adverse Events

All adverse events according to Common Terminology Criteria for Adverse Events v4.0. within 7 days (potential procedural related complications) after CAR T-cell infusion were reported.

2.7. Comparison Metrics of Infusion Method

To assess if CAR T cells injected in the tumor or the pleural cavity can be detected in systemic circulation, peak vector copy numbers—a quantitative real-time polymerase chain reaction assay was used to identify and quantify the presence of CAR T cells in the peripheral blood (vector copies per milliliter)—within one month following CAR T-cell infusion were compared between the two intrapleural infusion methods, intracavitary or intratumoral. In addition, metrics that reflect CAR T-cell activity in the immediate infusion period: fever, C-reactive protein (CRP), and interleukin-6 (IL-6) peak serum levels within 1 week following CAR T-cell infusion were compared between all patients that received regional infusion of CAR T cells (total number of procedures = 45), either by an indwelling pleural catheter placed by thoracic surgery or intervention pulmonary (number of procedures = 12) or by image-guided IR (number of procedures = 33).

2.8. Statistical Analyses

Quantitative variables were described as medians (minimum–maximum) and categorical variables as numbers (proportions, percentages). Normal distribution was tested with the Shapiro-Wilk test. Comparisons were performed using Fisher’s test, unpaired t-test and Mann-Whitney test for proportions, normal and non-normal distributed variables, respectively. Statistical analysis was performed using SPSS (SPSS^®, IBM^® SPSS^® Statistics, version 26, release 26.0.0.1, Armonk, New York). A P value < 0.05 was considered statistically significant.

3. RESULTS

3.1. Study Participant Characteristics

Thirty-one patients underwent an image-guided infusion among 41 total study participants in the study (Table 1). Two patients (Patients 19 and 24) had repeat image-guided infusions performed 8 and 8.4 months apart respectively (second procedures indicated as Patient 19* and 24* among 33 total number of procedures). Tumor diagnosis at the site of planned administration was confirmed by on-site histology and consisted of MPM for all patients except 1 patient (1/31, 3%) who had pleural metastases from breast cancer. The most common tumoral presentations were pleural thickening and pleural nodules and/or masses, with a mean diameter of 45 mm ± 28. Small loculated pleural effusion was present in 14 (14/33, 42%) patients. Eleven (11/31, 35%) patients underwent prior pleural fusion-promoting procedures—three (3/31, 10%) patients previously treated with radiotherapy to the hemithorax, 1 (1/31, 3%) patient with pleurectomy and decortication, 5 (5/31, 16%) patients with both, and 2 (2/31, 6%) patients (Patients 11* and 12*) had a previous infusion of CAR T cells through an indwelling pleural catheter.

Table 1.

Population and tumor baseline characteristics.

Characteristic	Total (n=31)
Age, years	70 (44–77)
Sex
Female	9 (9/31, 29%)
Male	22 (22/31, 71%)
Body mass index, kg/m2	27 (18–44)
Primary cancer
Mesothelioma	30 (30/31, 97%)
Breast	1 (1/31, 3%)
Pleural disease presentation
Multiple nodules/masses	22 (22/33, 67%)
Single nodule/mass	2 (2/33, 6%)
Nodule largest diameter, mm	40 (16–125)
Pleural thickening	27 (27/33, 82%)
Pleural effusion	14 (14/33, 42%)
Prior ipsilateral thoracic pleural fusion promoting treatments	11 (11/31, 35%)
Surgery	1 (1/31, 3%)
Radiotherapy	3 (3/31, 10%)
Surgery and radiotherapy	5 (5/31, 16%)
Interval duration from surgery to infusion, months	30 (10–41)
Interval duration from radiotherapy to infusion, months	22 (0.5–33)

Continuous variables are presented in median (minimum-maximum). Categorical variables are presented in raw numbers (proportions, percentages). Total number of patients= 31, total number of procedures= 33. Since two patients had a second image-guided infusion of CAR T cells, pleural disease presentation was considered for each procedure.

3.2. Selected Targets and Successful Injection Routes

Technical success was achieved in 100% of the cases, with 14 (14/33, 42%) infusions instilled in the pleural cavity and 19 (19/33, 58%) infusions instilled directly in the pleural tumor and/or peritumor. Successful infusion route and technical details for each procedure are presented in Figure 2.

Figure 2. Summarized technical details for image guided regional delivery of CAR T cells.

Bar chart showing injected volume, type of target and material used for each procedure. Cases of pleural expansion were noted.

In the intracavitary subgroup, 2 patients (Patients 19* and 24*) had a previous intratumoral infusion and 1 patient (Patient 12*) had a previous intracavitary infusion through an indwelling pleural catheter (Fig. 2). Infusions were administered into loculations in 11 (11/14, 79%) cases and into the pleural cavity after a successful pleural expansion in 1 (1/14, 7%) case. The 5 (5/6, 83%) other pleural expansions failed due to dense adhesions, among whom 2 patients had a prior history of ipsilateral radiotherapy and/or surgery to the pleura (Patients 25 and 41), but none were previously treated with infusion of CAR T cells in the pleural cavity. Despite failure of pleural expansion, the IR physician succeeded to place a 21G micropuncture needle between the pleura and the diaphragm and to inject complete volume of CAR T cells in 2 (2/14, 14%) cases (Patients 6 and 41).

In the intratumoral infusions subgroup, 1 patient (Patient 11*) had a previous ipsilateral intracavitary infusion through an indwelling pleural catheter. The target was a pleural nodule/mass in 13 (13/19, 68%) procedures, measuring 40 mm (23–125) and a pleural thickness in 6 (6/19, 32%) procedures, measuring 11 mm (6–17).

3.3. Imaging Guidance and Injection Material

In the intracavitary infusions subgroup, CT guidance was used in all cases, with additional ultrasound guidance in 6 (6/14, 43%) cases. Injections were performed through a 17G needle (3/14, 22%), 19G needle (4/14, 29%), 21G micropuncture set needle (2/14, 14%), 5Fr Yueh needle (2/14, 14%), 6Fr pigtail catheter (1/14, 7%), 8Fr Dawson-Mueller catheter (2/14, 14%).

In the intratumoral infusions subgroup, only CT guidance was used. Injections were performed through a 17G needle (14/19, 74%), 19G needle (2/19, 11%), 5Fr Yueh needle (1/19, 5%), 6Fr catheter (1/19, 5%) and 6.3Fr Dawson-Mueller catheter (1/19, 5%).

There was no statistically significant difference between intracavitary and intratumoral infusions of injected volumes (114 mL [10–176] vs. 65 mL [30–186], P= 0.3) and doses (60M [0.3M-60M] vs. 30M [0.3M-60M], P= 0.3) (Table 2), but anesthesia time was lower for intratumoral injections, measured at 55 minutes (25–107) vs. 70 minutes (45–120) in the intracavitary group (P= 0.03).

Table 2.

Comparison of intratumoral and intracavitary infusion routes of CAR T cells.

Variable	Image guided infusion by IR		P
	Intratumoral (N= 19)	Intracavitary (N= 14)
Volume injected, mL	65 (30–186)	114 (10–176)	0.3
Dose, cells/Kg	30M (0.3M–60M)	60M (0.3M–60M)	0.3
Peak vector copy numberb, copies/mL	3340 (0–77300)	5895 (438–21960)	0.3
Fevera	4 (4/19, 21%)	4 (4/14, 29%)	0.7
Peak IL-6a, pg/mL	113 (13–949)	103 (5–3800)	1
Peak CRPa, mg/dL	14 (1–29)	16 (2–33)	0.6

Continuous variables are presented in median (minimum-maximum). Categorical variables are presented in raw numbers (proportions, percentages). CAR= chimeric antigen receptor; CRP= C-reactive protein; IL= interleukin; IR= interventional radiology.

^aWithin 0–7 days of infusion.

^bWithin 0–30 days of infusion.

3.4. Procedural Safety and Post-procedure Image Characteristics

No adverse events > grade 1 related to the procedure (needle or catheter placement) were observed. Post-infusion imaging findings are summarized in Figure 3. The most common were moderate ground glass opacities associated with interlobular septal thickening and/or consolidation (Fig. 3B, 3E), observed more frequently after intratumoral than after intracavitary infusions (10 [10/19, 53%] vs. 2 [2/14, 14%], P= 0.02) (Fig. 3H). These changes occurred in the parenchyma adjacent to the injection site and were of mild to moderate extension, except in 1 case where they were diffuse (Fig. 3B, Patient 25). In this case, the patient did not have any noticeable clinical findings such as chest pain/discomfort, cough, or change in oxygen saturation, and the planned infusion was continued. Two patients (2/33, 6%) had small asymptomatic pneumothoraxes (grade 1) that were stable on 48-hour follow-up imaging and did not require chest tube placement or prolonged hospital stay. No hemoptysis or hemothorax were observed.

Figure 3. Intra- and post-procedural imaging characteristics.

(A) Attempted pleural expansion in Patient 25 who had prior pleurectomy decortication and radiation therapy with a 19G needle (short arrow) to facilitate pleural infusion of CAR T cells was not successful. Air administered through the needle tracked superiorly and along the right pectoralis muscle (long arrow). Thus, infusion was performed directly into a pleural nodule (arrowhead). (B) In this same patient, increasing ground glass opacities and interlobular septal thickening (arrowhead) were observed on intraprocedural non contrast chest axial CT scan. Note the absence of these parenchymal findings before the infusion, as shown in A. The patient respiratory status was stable throughout without cough or change in oxygen saturation suggesting intralymphatic/visceral pleural administration. Following successful administration of planned volume, the 19G needle was then removed and post procedure radiographs showed stability of the small right pneumothorax (long arrow), not requiring any chest tube placement. (C) Axial CT scan of the chest performed 3 weeks after CAR T cells infusion showed complete resolution of previous imaging findings. (D) In another example, pre-procedural CT scan of the chest (Patient 19*) performed 5 days before the procedure showed normal lung parenchyma in the right lower lobe. (E) Mild ground glass opacities and interlobular septal thickening (arrowhead) occurred after pleural injection of CAR T cells. (F) CT of the chest performed 1 month after CAR T cells infusion showed near complete resolution of previous imaging findings. (G) Slight extravasation of infusate below the diaphragm (long arrows) occurred in one patient (Patient 41), without associated clinical symptoms. (H) Table summarizing per procedural and post infusion imaging findings. Ground glass opacities and interlobular septal thickening were mild to moderate in all cases except in Patient 25 where they were diffuse.

IR= interventional radiology

3.5. CAR T-cell Infusion Metrics: Intracavitary vs. Intratumoral Infusions

Only 2 (2/33, 6%) serious adverse events (grade ≥ 3) consisting of a cytokine release syndrome and dyspnea occurred between the intratumoral and intracavitary groups within 7 days following CAR T-cell infusions. All non-biological adverse events within 7 days following CAR T-cell infusions are listed in Supplementary Table 1.

Fever within 7 days of infusion was observed in 8 (8/33, 24%) procedures, with no statistically significant difference between intratumoral and intracavitary infusions reported (4 [4/19, 21%] vs. 4 [4/14, 29%], P= 0.7). Similarly, there was no statistically significant difference between intratumoral and intracavitary injections regarding peak IL-6 (113 pg/mL [13–949] vs. 103 pg/mL [5–3800], P= 1) and CRP levels (14 mg/dL [1–29] vs. 16 mg/dL [2–33], P= 0.6) reported within 7 days after CAR T-cell infusion. Peak vector copy number within 30 days after CAR T-cell infusion reached 3340 copies/mL (0–77300) in the intratumoral group and 5895 copies/mL (438–21960) in the intracavitary group, and the difference was not statistically significant (P= 0.3) (Table 2).

No statistically significant difference was reported between patients that received CAR T-cell infusion by interventional radiology or through an indwelling pleural catheter placed by thoracic surgery or intervention pulmonary procedures regarding fever (8 [8/33, 24%] vs. 1 [1/12, 8%], P= 0.3), peak IL-6 (113 pg/mL [5–3800] vs. 58 pg/mL [24–305], P= 0.5) and CRP levels (15 mg/dL [1–33] vs. 10 mg/dL [0–26], P= 0.06) within 7 days after CAR T-cell infusion.

4. DISCUSSION

Our results from a larger series of pleural cancer patients demonstrate that image-guided regional infusion of CAR T cells in the setting of a phase I/II clinical trial was successful and safe. Using two infusion routes, intracavitary and intratumoral, delivery of CAR T cells was achieved across all tumoral presentations of pleural malignancies, including patients previously treated with radiotherapy to the pleura, surgery, or both in ipsilateral hemithorax. Our study also supports the repeatability of regional infusion of CAR T cells. We have previously demonstrated the benefit of performing correlative immune analyses of pleural fluid and pleural biopsies in comparison with systemic immune factors [19].

The first considerations for a novel approach to deliver immunotherapeutics are technical feasibility and safety considerations [26]. As demonstrated in our series, patients with pleural malignancies may lack pleural effusion or present with a fused cavity from prior therapy that prevent access to the pleural space [34]. With improved imaging methods and techniques, combined with experience garnered from difficult intrathoracic diagnostic (multiple core biopsies) and therapeutic (catheter insertion, embolization, and ablation) procedures, we were able to successfully perform IR-guided delivery of even a large volume (> 150 ml) of therapeutic agent within the stipulated time frame. A successful delivery was achieved either by artificially expanding the pleural space or by directly injecting CAR T cells in the pleural nodules/masses or the pleural thickening. While all infusions were safe and successful when performed by experienced IR physicians, in 5 patients with prior pleural fusion procedures, an alternative approach was required. This is an important consideration in planning as there is only a 3-hour time limit to inject cells with a high manufacturing cost per patient. Consolidative or ground glass appearance and interlobular septal thickening can be observed after local infusion in up to 36% of the procedures, more often after intratumoral than intracavitary delivery, perhaps related to the tumor lymphatic drainage and spread of the instilled fluid in the tumor [39]. Patients with these imaging findings remained asymptomatic and did not require any intervention even when the pulmonary image findings were diffuse; the operator should be aware of these radiological changes which in our cohort were asymptomatic. Our preclinical studies show that CAR T cells when inadvertently administered into subcutaneous tissue or intramuscular or peritoneal cavity, are absorbed into systemic circulation and traffic to tumor, albeit with decreased efficacy. However, to assess safety and efficacy, there is need for uniform dose delivery within a cohort. Building upon our experience, we provide a flowchart to standardize delivery route and target selection in Figure 4.

Figure 4. Algorithmic approach for optimal CAR T-cell infusion route selection.

Unlike the intraventricular infusion of CAR T cells that resulted in the regression of recurrent multifocal glioblastoma including spinal metastases, wherein CAR T cells remained within the central nervous system due to the blood-brain barrier [16], CAR T cells delivered regionally either in the pleural cavitary or locally in the tumor both reached systemic circulation with no difference in peak vector copy numbers between infusion routes. These results are in line with our pre-clinical study that showed that regionally delivered CAR T cells, irrespective of site of administration, can not only infiltrate the diffuse pleural tumor but also circulate and efficiently traffic to distant tumor sites [4]. In the presence of diffuse tumor or multiple pleural nodules, in our study, target selection was based on lesion size and uptake on recent PET CT, as lesions size ≤ 2 cm has been associated with increased safety in CT-guided percutaneous lung biopsies [40]. Uptake on recent PET CT was also considered when available to target a metabolically active part of the tumor [41].

Image-guided interventional procedures have revolutionized treatments in the field of cardiology, cardiac surgery, vascular surgery, and neurosurgery. While applying the advantages of IR, the critical component of our approach is based on the strong clinical, biological, and immunological rationale developed from preclinical studies in clinically relevant mouse models. Regional aggressive disease with relatively uncommon distant metastases provides the clinical rationale to treat MPM with regional delivery. Novel genetic engineering adopted in the development of our CAR construct to target high mesothelin-expressing tumor and spare adjacent low mesothelin-expressing normal pleura and pericardium supports the biological rationale. In addition to avoiding pulmonary sequestration that occurs following systemic delivery; immediate cancer-antigen activation with ensuing proliferation and dose expansion of intrapleurally delivered CAR T cells provides the immunological basis. Our unpublished data and emerging published data from others demonstrate that regionally delivered and antigen-activated CAR T cells not only circulate systemically but also provide a ‘swarming effect’ to attract other effector T cells to the tumor aided by a chemokine gradient, and they themselves can enter distant metastases in an improved fashion due to upregulated chemokine receptors [42]. Unlike in intraperitoneal administration where the delivery and subsequent inflammatory response can precipitate bowel obstruction or perforation, intrapleural delivery in our study did not result in any procedure-related morbidity while successfully eliminating pulmonary toxicity. Published evidence of augmenting systemic immunity following regional administration of oncolytic viruses or regional administration of immune checkpoint inhibitors agents into the pleural cavity effectively providing checkpoint blockade in tumor draining lymph nodes attests to the expansion of the applicability of regional immunotherapy to augment systemic immunity [11,43,44].

While our study used standard introducer needles or catheters to deliver CAR T cells, Muñoz et al. showed in their preclinical work that multiside hole needles allowed better intratumoral distribution and retention compared to end hole needles, along with decreasing the pressure during the injection [45]. More interestingly, an increase of type I interferon-associated genes following multiside hole needles injections suggested a potential influence of the injection technique on the drug efficacy [45]. A standardized administrative method, technique, and access equipment is preferrable to ensure uniform instillation of therapeutic agents [24]. With the aim of ensuring a homogeneous intratumoral distribution, Tselikas et al. suggested to target different parts of the tumor, either during the same procedure (“radial technique”) or through repeated injections over time (“sequential technique”), although these approaches seem more technically challenging and might increase the procedure related complications, particularly for tumors presenting with minimal pleural thickening [26]. In the future, repurposing port-a-cath devices used for repeated intravenous injections and development of multiside hole needles that could be placed in the pleural tumor and attached to a subcutaneous device might facilitate repeated administration as well as sampling.

There are several limitations in our study. First, the infused agent was not visible on conventional imaging, which prevented the operator from assessing the drug’s distribution after the injection. However, CT was used in every procedure to confirm the needle or catheter position in the target before injection. Radiopaque drug delivery vehicles, like ethiodized oil used for liver trans-arterial chemoembolizations [46], could allow an accurate intra- and post-procedural assessment of intra- and peritumoral distribution and could also serve to tag the tumor for imaging follow-up. However, addition of such agents to therapeutic agent requires preclinical and pharmacokinetic studies to ensure no compromise of CAR T-cell viability and function; pre-clinical studies are underway in our laboratory [7]. Second, a multi-center study with larger series of patients is necessary to validate our approach. Finally, this study only evaluated mesothelin-targeted CAR T cells; given the role of the specificity of the targeted antigen in limiting on-target off-tumor reactions [47], our short-term results in an early-phase study cannot be extrapolated to other immunotherapeutic agents.

4.1. Conclusions

In conclusion, image-guided regional infusion of CAR T cells for pleural malignancies is successful, safe, and repeatable. We described CAR T-cell infusion in the pleural cavity or directly in the pleural tumor, with a feasibility across all anatomical variations in tumoral presentation. An algorithmic approach for selection of infusion route, target and procedural instruments and techniques is key for optimal efficacy and consistency of outcomes among regional delivery of immunotherapy trials.

Highlights:

• CAR T cells administered intrapleurally in 31 participants with pleural cancers

• Intrapleural delivery of CAR T cells using intracavitary/intratumoral routes is safe

• Repeated administration of therapeutic agents to the pleural cavity is feasible