Phase II trial of Pembrolizumab and Anti-CD3 x Anti-HER2 Bispecific Antibody Armed Activated T Cells in Metastatic Castrate Resistant Prostate Cancer

Abstract

Purpose:

A phase II study was conducted to evaluate the safety and efficacy of the combination of HER2 BATs and programmed death −1 (PD-1) inhibitor, pembrolizumab.

Patients and Methods:

Metastatic castrate resistant prostate cancer (mCRPC) patients with 0–1 performance status (PS) and normal liver, kidney and marrow function, pre or post docetaxel chemotherapy were eligible. Primary endpoint was 6 month progression free survival. Peripheral blood mononuclear cells (PBMC) were obtained by a single apheresis, shipped to University of Virginia, activated with OKT3 and expanded for 14 days in IL-2, harvested, and armed with HER2Bi and cryopreserved. HER2 BATs were infused twice weekly for 4 weeks and pembrolizumab was administered every 21 days for a maximum duration of 6 months starting 1–3 weeks prior to HER2 BATs infusion.

Results:

Fourteen patients were enrolled with a median age of 69 (range 57–82 years) and median PSA of 143.4 (range 8.2 – 4210 ng/dL). Two patients had peritoneal metastases, 1 had lymph node (LN) only metastases and 11 had bone metastases of which 7 had bone and LN metastases. All were pretreated with androgen receptor axis targeted agents and 7 (50%) had prior docetaxel chemotherapy. The toxicities were grade1–2 infusion reactions with fever, chills, headaches, nausea and/or myalgias. Primary endpoint of 6 month progression free survival (PFS) was achieved in 5 of 14 patients (38.5%, 95%CI 19.5%–76.5%). Median PFS was 5 months and median survival was 31.6 months.

Conclusions:

The safety and promising efficacy makes this combination worthy of future investigation in mCRPC.

INTRODUCTION

Despite recent advances in treatment options, metastatic castrate resistant prostate cancer (mCRPC) remains an incurable malignancy. Although cellular therapies have gathered momentum in mCRPC, the clinical impact of sipuleucel T, which is the only approved agent, has been modest [1]. Novel therapies are needed to induce remissions, decrease morbidity, and improve survival in mCRPC. Multiple bispecific antibodies and cellular therapies are under active investigation in mCRPC [2].

Since HER2 is overexpressed in 42–70% of mCRPC cases, treating patients with trastuzumab was investigated but minimal efficacy was noted [3,4]. Treating mCRPC patients, using ex vivo expanded anti-CD3 activated T cells (ATC) [5–9] armed with anti-CD3 x anti-Her2 bispecific antibody (HER2Bi), that kill HER2 targets in a non-MHC restricted manner, may fulfill this unmet clinical need [10,11].

HER2Bi armed ATC [HER2 BATS] have been shown to proliferate and have demonstrated cytotoxicity and trafficking to tumors, and secretion of cytokines such as IFNγ, TNFα, and GM-CSF, upon tumor engagement [10,11,12]. Preclinical studies show that HER2 BATs bind and kill low HER2 expressing PC-3 cell line as well as DU-145, and LNCaP cell lines [12, 13]. In a phase I trial in 7 patients with mCRPC, there were no dose limiting toxicities (DLT) and 3 of 7 patients had decreased prostate-specific antigen (PSA) levels and relief of bone pain [14].

The combination of anti-CTLA-4 mAb (ipilimumab) checkpoint inhibitor (CI) enhanced in vitro T cell proliferation and cytotoxicity [15]. Expansion of ATC in the presence of ipilimumab significantly improved both T cell proliferation and enhanced bispecific antibody redirected specific cytotoxicity of ATC [15]. We considered a study design of combination with ipilimumab, a CTLA-4 inhibitor, however the toxicity of this agent was a limiting factor in an elderly pretreated patient population, and the randomized clinical trial in mCRPC of RT +/− ipilimumab did not demonstrate benefit [16, 17]. There were nine treatment related deaths in the ipilimumab arm of the study. Given the tolerability of PD- 1 inhibitor and preliminary results of efficacy in mCRPC, this was selected as the immune checkpoint therapy for the study [18].

A phase I study using HER2Bi armed ATC (HER2 BATs) in metastatic breast cancer (MBC) showed induction of immune responses and encouraging survival results with a median OS of 37 months [19]. A phase II trial in heavily pretreated 24 HER2 negative and hormone receptor (HR) positive, and 8 triple negative breast cancer (TNBC) patients showed a median OS of 13.1 (95% CI 8.6 to 17.4), 15.2 (95% CI 8.6 to 19.8), and 12.3 (95% CI 2.1 to 17.8) months for the entire group, HER2-ve HR+, and TNBC patients, respectively [20]. Phase I/II metastatic breast cancer and mCRPC trials showed that infusions of HER2 BATs induced cytotoxic T cell activity, mediated by fresh PBMC, directed at breast or prostate cancer cell lines, are capable of inducing immune responses that translate into clinical responses [19, 20]. Preclinical studies showed that HER2 or EGFR BATs can target cells with very low tumor antigen expression. HER2 BATs exhibit high levels of cytotoxicity against the MCF-7 cells which is a HER2 negative breast cancer cell line by immunohistochemistry [21] It is clear that only a few molecules of target antigens which may or may not be detectable by flow cytometry or immunohistochemistry are sufficient for target binding and mediation of non-MHC restricted cytotoxicity.

The preclinical studies of combining checkpoint inhibitor with BATs and our phase I clinical trial results of BATs in mCRPC, provided a strong rationale for planning a phase II trial. This study hypothesized that the combination of 8 weekly infusions of HER2 BATs with every 3 week infusions of pembrolizumab would result in a favorable progression free interval in men with mCRPC.

PATIENTS AND METHODS

Study Design.

The primary objective of this study was to evaluate the proportion of patients that remained progression free for a duration of 6 months or longer. The study was approved by the Wayne State University and University of Virginia institutional review board and written informed consent was obtained from all patients before registration. The study was conducted in accordance with recognized ethical guidelines per the Declaration of Helsinki for medical research involving human subjects. Dr. L.G. Lum sponsors the Investigational New Drug Application BB-#9985 that supports the production of HER2 BATs and this protocol. The clinical efficacy of 8 infusions of HER2 BATs (up to 10¹⁰/infusion) given twice per week for 4 weeks in combination with anti-PD-1 therapy pembrolizumab was evaluated. Pembrolizumab was administered once every 3 weeks starting 1– 3 weeks before the 1st BATs infusion. The secondary objectives for the combination immunotherapy were to assess the safety, response rates, and to estimate OS. The exploratory objectives consisted of magnitude of change in anti-PC immune functions and cell markers after HER2 BATs infusions, the type, number, and density of tumor infiltrating lymphocytes and PD-1 expression in pretreatment biopsies, and the serum Th₁/Th₂ ratio in prostate cancer tumor tissue at baseline and after immunotherapy.

Patient selection.

Eligible patients were 18 years or older with histologically confirmed prostate adenocarcinoma with radiologic evidence of metastases. Progression was required by either PSA, RECIST 1.1 criteria for measurable disease or new areas of metastases on bone scan. Castrate level of testosterone (Level<50 ng/ml) was required. A minimum duration of two weeks since any immunosuppressive therapy or prior androgen axis targeting agents, and four weeks since prior chemotherapy were required. The patient had to agree to use an adequate method of contraception starting with the first dose of study therapy through 120 days after the last dose of study therapy. Patients with performance status ≤ 2 and life expectancy of 6 months or more were eligible. Patients were required to have adequate bone marrow, liver, and renal function.

Production of anti-CD3 x anti-HER2 BiAb (Her2Bi).

Her2Bi was manufactured by heteroconjugation of murine IgG2a anti-CD3 muromonab (OKT3, Miltenyi Biotec, Auburn, CA) and humanized anti-HER2 IgG₁ (Herceptin^®, Genentech Inc., South San Francisco, CA) using sulpho-succinimidyl 4-(N-Maleimidomethyl) cyclohexane-1-carboxylate (Sulpho-SMCC) and Traut’s reagents [21].

Treatment Plan.

Pheresis product collected from the patients’ was activated with 20 ng/ml of OKT3 and expanded in 100 IU/ml of IL-2 for 14 days in RPMI-1640 supplemented with 10% FBS as previously described[1]. Activated T cells (ATC) were harvested and armed with anti-CD3 x anti-HER2 bispecific antibody [HER2Bi]) at a pre-optimized concentration of 50 ng/10⁶ ATC as previously described[1]. Armed ATC were washed twice to remove unbound HER2Bi and cryopreserved in 8 equal aliquots until they are thawed at the bedside. Arming ATC with HER2 BiAb (HER2 BATs) converts every T cell into a non-MHC restricted HER2-specific cytotoxic T lymphocyte (CTL). HER2 BATs have been shown to repeatedly kill, proliferate, and release of several cytokines that induce DC maturation. The HER2Bi armed ATC (HER2 BATs) were tested for specific cytotoxicity, immune subsets, pathogens, mycoplasma, and endotoxin prior to release for clinical infusion. Pembrolizumab was administered at a flat dose of 200 mg intravenously over about 30 minutes every 21 days for a maximum of 9 doses. The dose and schedule of therapy is depicted in Figure 1.

Figure 1.

A) Treatment Schema showing schedule of infusions, immune evaluations, and tumor evaluations, B) BATs manufacturing schema

Phenotyping of Cell Therapy Product (BATs).

The harvested ATC product was stained for CD3⁺, CD4⁺, CD8⁺, CD25⁺, CD19⁺, CD20⁺, CD45RO⁺, CD45RA⁺, CD19⁺, CD20⁺, CD56⁺, CD127⁺, CD11b⁺, CD33⁺, and HLA-DR⁺ cells by flow cytometry and analyzed for T effector cells, T effector memory cells, T memory cells, T central memory cells, NK cells, B cells, Tregs, and myeloid derived suppressor cells. Cryopreserved PBMC from the time of leukapheresis served as baseline controls.

Functional Activity of Cell Therapy Product for Specific Cytotoxicity.

PC-3 target cells were plated in 96-well flat-bottom microtiter plates at 4×10⁴ cells/well, allowed to adhere overnight at 37°C, and labeled with ⁵¹Cr at 20 (μCi/mL) in the labeling media (50% fetal bovine serum (FBS) in RPMI-1640 supplemented with 10% fetal bovine serum, 2% penicillin-streptomycin, and 1% L-glutamine. [21]. Unarmed ATC and HER2 BATs were added at an effector: target (E:T) ratio of 25:1. Co-cultures were incubated for 18 hours and the supernatants were counted to quantitate ⁵¹Cr release with % specific cytotoxicity = (experimental counts per minute (cpm) − spontaneous cpm) / (maximum cpm − spontaneous cpm) × 100. Means and standard errors were calculated from four to six replicates per sample.

Immune Monitoring Studies

Specific IFN-γ EliSpots.

Specific CTL activity was evaluated by stimulating fresh PBMC from patients with (PC cell line, PC3) or K562 cells (NK cell target) to measure interferon gamma (IFN-γ) Elispots to a surrogate measure of CD8-mediated memory CTL activity and CD4-mediated T helper responses [22]. IFN-γ EliSpots produced by PBMC were assessed after 18 hours of stimulation with PC3 or K562 after subtracting spontaneous IFN-γ EliSpots produced by PBMC without any stimulation at an effector to target ratio (E/T) of 1:1 as previously described.

Real Time Cytotoxicity Assay.

In the Real Time Cell Analysis (RTCA) using xCELLigence system, cytotoxicity was measured by impedance readout as Cell Index (CI) to monitor real-time changes in cell number. Cell attachment was monitored using the RTCA software until the plateau phase was reached, which was usually after approximately 22–24 hours before adding effector cells [21]. Prostate cancer line PC3 was plated at an optimal of 20,000 cells/well in 96-well E-Plates and allowed to adhere until reaching the CI of 1.0. PBMC effectors at E:T of 2:1 or 1:1 were added and PC-3 impedance signals were continuously monitored for 72–120 hours. Untreated targets or effectors alone served as controls. CI values were exported and % lysis was calculated in relation to the change in tumor cell impedance without effector cells.

Phenotyping of Pre and Post Therapy PBMC.

The harvested ATC product was phenotyped for CD3⁺, CD4⁺, CD8⁺, CD25⁺, CD19⁺, CD20⁺, CD45RO⁺, CD45RA⁺, CD19⁺, CD20⁺, CD56⁺, CD127⁺, CD11b⁺, CD33⁺, and HLA-DR⁺ cells by flow cytometry and analyzed for T effector cells, T effector memory cells, T memory cells, T central memory cells, NK cells, B cells, Tregs and myeloid derived suppressor cells. Cryopreserved PBMC from the time of leukapheresis served as baseline controls.

Serum Cytokines.

Cytokines and chemokines were measured in the serum at selected time points using a 25-plex human cytokine Luminex Array (Invitrogen, Carlsbad, CA) using BioPlex system (Bio-Rad Lab., Hercules, CA). The multiplex panel included IL-1β, IL-1 receptor antagonist (IL-1RA), IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-13, IL-17, tumor necrosis factor (TNF-α), interferon-alpha (IFN-α), IFN-γ, GM-CSF, macrophage inhibitory protein (MIP-1α), MIP-1β, MIP-3β, IFN-γ-induced protein-10 (IP-10), RANTES and monocyte chemotactic protein (MCP)-1. This assay was able to detect < 10 pg/mL. The cytokine levels were calculated from a standard curve [22].

Staining for Vβ Repertoire by Multicolor Flow Cytometry.

Individual Vβ clones were detected in PBMC using TCR-Vβ repertoire kit from Beckman Coulter as per manufacturer’s instructions. Cells were stained simultaneously with for CD3 and IFN-γ, and a set of three antibodies directed against TCR-Vβ repertoires and analyzed by flow cytometry. Cells were gated on CD3⁺cells, individual TCR families were then analyzed for IFN-γ positive CD3⁺ T cells.

Staining for HER2 BATs in the peripheral blood. The percentage of anti-CD3 x anti-HER2 positive cells was quantitated by using goat PE conjugated anti-mouse IgG2a antibodies to detect OKT3 in the BiAb as reported previously [21].

Biopsy Tissue Procurement and Analysis.

Metastatic image guided biopsy collection was performed by interventional radiology. From each patient 1–3 cores were collected in 10% PBS buffered formaldehyde solution. Formalin fixed bone biopsy tissues were decalcified in 10 % EDTA solution prior to embedding in paraffin, whereas lymph node biopsy tissues were embedded subsequent to formalin fixation.

Immunohistochemical Staining.

Tissue sections were stained with monoclonal antibodies recognizing CD4, CD8, CD20 (B-cells), CD68 (Macrophages) and FoxP3 (Tregs) for immune cell population. Antibodies recognizing IFN-γ and IL-10 were used to determine Th₁ and Th₂ responses. Isotype-matched mouse monoclonal antibodies was used as negative control. Slides were stained with anti-CD4/anri-CD8/anti-CD19/anti-CD68/antiFoxP3 antibody cocktail, using automated protocol on a Ventana Discovery Ultra instrument (Roche Diagnostics, Ventana Medical Systems, Tucson, AZ). Sections of human tonsils known to contain abundant various immune cell populations were used as positive controls. Slides were scanned on Hamamatsu slide scanner NanoZoomer S360 and images were viewed on the NDP viewer software. Analysis of images were performed on online Visiopharm software, this software is run on a server supported by the UVA ITS. Dr. Christopher Moskaluk (Chair of Pathology Professor of Biochemistry & Molecular Genetics, University of Virginia) marked the region of interest ‘ROI’s’ to identify cancerous regions. Staining, scanning and image analysis were done by the UVA Biorepository & Tissue Research Facility. Qualitative and quantitative measurements of tumor infiltrating mononuclear cells were correlated with clinical outcomes.

Statistical Analysis.

A single arm two-stage design was planned. The primary objective was to assess the clinical efficacy of the combination of HER2BATS and pembrolizumab in mCRPC. The primary endpoint was the proportion of patients achieving clinical progression-free interval of 6 months from study registration. The clinical progression was defined by PCWG II criteria and was assessed by clinical evaluation, monthly PSA monitoring and radiologic assessment at 3 and 6 months, and every 6-month interval thereafter.

The study used Simon’s two-stage design to test the null hypothesis of 20% or less versus the alternative hypothesis of more than 20% patients achieving progression free time of ≥ 6 months. With 5% Type I error and 80% power, the Simon’s two-stage minmax design required a total of 33 patients and had a probability of early termination of 0.716 if the treatment is not promising. The PASS12 was used for sample size calculation [23,24]

At the first stage, 18 evaluable patients were to be enrolled and the trial would proceed to second stage. If more than 4 patients were progression free at 6 months, 15 more patients would be enrolled. Due to COVID-19 pandemic cellular therapies were suspended and hence the study was not completed. However, follow up revealed that >20% patients were progression free at 6 months and the requirements for proceeding to second stage were met.

Immune responses at post BATs infusions (postIT) were compared with pre-study IFN-γ Elispots, specific cytotoxicity, cytokine/chemokine profiles. These were analyzed using Wilcoxon signed-rank test. Due to early termination of the trial, the study sample size was smaller than the designed sample size. All correlative studies are descriptive and exploratory. All p values reported are raw p values not adjusted for multiple testing.

Data Availability Statement:

The original contributions presented in the study are included in the article/ Supplementary Material. Further inquiries can be directed to the corresponding authors.

RESULTS

Clinical Status.

Total 14 patients were enrolled with a median age of 69 years (range 57–82 years) and median PSA of 143.4 ng/dL (range 8.2 – 4210.3 ng/dL). Two patients had peritoneal metastases, one had lymph node (LN) only metastasis and 7 of 11 with bone metastases also had LN metastases (Table 1). The representativeness of the population is discussed in Table S1. All had received prior androgen receptor axis targeted agents and 7 had prior chemotherapy. Four patients (28.5%) completed the entire course of combination immunotherapy and 10 patients (71.4%) received all infusions of HER2 BATs. The HER2 BATs associated toxicities consisted mainly of grade 1– 2 infusion reactions with fever, chills, headaches, nausea and/or myalgias (Table 2).

Table 1.

Patient Characteristics

Patient Characteristics	Frequency N= 14
Age (median, range)	69 years (57–82 years)
Baseline PSA (median, range)	143.4 (range 8.2 – 4210.3 ng/dL)
Race AA/CA	1 (7% ) / 13 (93%)
Gleason score 6/7/8–10/unknown	1 (7%)/ 3 (21%) /9 (65%)/1 (7%)
Performance Status:
Zubrod score 0	5 (36%)
Zubrod score 1	9 (64%)
Prior Therapy
Abiraterone/ enzalutamide	4 (28.5%)
Both	9 (64.5%)
Radium-223	4 (28.5%)
Sipuleucel T	1 (7%)
Prior chemotherapy:
Docetaxel	7 (50%)
Cabazitaxel	2 (14%)
Both	2 (14%)
Sites of disease
Lymph node and bone	6 (43%)
Visceral: peritoneal (2) and adrenal (1)	3 (21%)
Bone only	4 (29%)
Lymph node only	1 (7%)

Table 2.

Treatment Related Toxicities

Toxicity	BATS related	Pembrolizumab related
Headache	Grade 1: 6 patients	None
Fever	Grade 1- 3 patients	None
Chills	Grade 1- 6 patients Grade 2 – 3 patients	None
Fatigue	Grade 1- 4 patients Grade 2- 2 patients Grade 3- 2 patients	Grade 3- 2 patients
Myalgias	Grade 2- 2 patients	Grade 2- 2 patients
Infusion reaction	Grade 1- 1 patient Grade 2- 1 patient	None
Mental status change	Grade 3- 1 patient	Grade 3- 1 patient

One patient died of progressive disease before starting therapy. Thirteen patients were evaluable for response. PSA response and duration of response are shown in Figures. 2A and B. Six patients demonstrated PSA decline of 25% or greater. Five patients (38.4%) were free of progression at 6 months. The results qualify the regimen for future investigation. Two patients progressed prior to completing 12 weeks of therapy. Median PFS was 5 months, 95% CI (4 months, Not reached). Median OS was 31.6 months, 95% CI (7.4 months, Not reached) [Figure. 2C]. At the time of last analysis, six patients are alive, of which five are alive for > 2 years and one is alive at 9 months follow up. Of 7 patients who died, 6 were due to progression of malignancy and one was an accidental death. Two patients progressed with visceral metastases and a low PSA clinically suggestive of neuroendocrine transformation. Four patients maintained a sustained response ranging from 7.5 months to 11.5 months.

Figure 2.

A) Waterfall plot of PSA change in evaluable patients B) Swimmers plot showing duration of response. C) Kaplan-Meier (K-M) survival curves showing overall survival (OS) and Progression free survival (PFS)

Phenotyping of Cell Product.

Viability, the proportion of CD3, CD4, and CD8, the CD4/CD8 ratio, and cytotoxic T cell activity by ⁵¹Cr release is summarized in Table S2. There were no correlations between the ATC phenotype, CD4/CD8 and cytotoxic T cell activity and clinical status of the patients. No correlation was detected between the various immune marker expressions and achievement of 6-month PFS endpoint.

Post Therapy Immune Monitoring Studies

Significantly Enhanced Specific Cytotoxic Activity in PBMC after Infusions of HER2 BATs.

Fresh PBMC from 12 patients were tested for cytotoxicity against PC-3 cells by RTCA using xCELLigence system at pre-therapy (PreIT), mid therapy, and post therapy time points (PostIT). Since each patient showed the peak immune response at different time points post therapy, we compared the highest post treatment cytotoxicity response with the preIT sample. Specific cytotoxicity by Post-IT PBMC was significantly higher (p<0.004) against PC-3 targets compared to the preIT baseline PBMC samples. Specific cytotoxicity by PostIT ranged between 17–50% compared to ~0–37% cytotoxicity at preIT baseline at E:T of 2:1 against PC-3 cells at 72 hours (Figure. 3, top panel).

Figure 3.

PBMC from all twelve PC patients were tested for direct cytotoxicity against PC-3 cell line at 2:1 effector to target ratio (E/T, 2:1). Upper panel shows the highest specific (PC-3) cytotoxicity postIT displayed as dot plot on left and line graph on the right. Specific cytotoxicity increased significantly (p<0.004) after multiple HER2 BATs infusions (PostIT) compared preIT cytotoxicity. Middle panel shows the PC-3 specific IFN-γ Elispots responses postIT compared to preIT base line responses and lower panel shows innate IFN-γ Elispots responses against NK target K562 cells.

Enhanced CTL and Natural Killer (NK) Activity in PBMC after Infusions of HER2 BATs.

Fresh PBMC from 12 patients were tested for specific anti-prostate cancer cytotoxicity and innate killer cell activity by measuring the IFNγ ELISpots upon exposure to PC-3 or K562, respectively, at pre-therapy (PreIT), during therapy, and post treatment time points (PostIT). There were enhanced IFN-γ responses above background seen in post-IT PBMC, but increases in the numbers of IFN-γ secreting T cells did not increase upon stimulation with PC-3 cells. (Fig. 3, middle panel). Similarly, there were increased levels of IFN-γ activity in post-IT PBMC, but the increases did not increase upon stimulation with NK specific K562 cells (Fig. 3, lower panel). Data is presented at the highest post treatment specific and NK activity compared with preIT sample.

Significant Decrease in Tregs after HER2 BATs infusions.

Phenotyping at pre- and postIT PBMC from 12 patients at the designated time points are shown in Figs. 4A and 4B. There were no significant differences observed for B cells, MDSC or any of the T cell subpopulations at postIT time points compared to preIT baseline except for Tregs. Phenotyping at pre- and post IT PBMC was performed for the proportion of CD4⁺ and CD8⁺ T cells, Naïve T cells (CCR7⁺/CD45RO⁻/CD45RA⁺⁾, T effector cells (Teff, CCR7⁻/CD45RO⁺/CD45RA⁻), T central memory cells (Tcm, CCR7⁺/CD45RO⁺/CD45RA⁻), T regulatory cells (Tregs, CD4⁺/CD25⁺/CD127⁻), B cells and MDSC ( CD11b⁺/CD33⁺/HLA-DR⁻). Tregs were significantly lower (p<0.01) after 4 infusions of BATs (pre inf 5 time point) compared to pre Inf 1 baseline time point (Figure. 4A, top right panel).

Figure 4.

A) Shows the phenotyping for CD3, CD4, CD8, Tregs, MDSC and B cells at pre- and postIT PBMC from 12 patients at the designated time points. No significant differences observed for T cell subsets, B-cells, MDSC at postIT time points compared to preIT baseline except for Tregs. Tregs were significantly lower (p<0.01) at pre infusion #5 time point postIT. B) Shows phenotyping for NK cells, Naïve T cells (CCR7⁺/CD45RO⁻/CD45RA⁺⁾, T effector cells (Teff, CCR7⁻/CD45RO⁺/CD45RA⁻), T central memory cells (Tcm, CCR7⁺/CD45RO⁺/CD45RA⁻), there were no differences observed for any memory T cell subsets or NK cells between pre- and postIT time points.

The Th₁/Th₂ cytokine and chemokine responses.

We used a 25-panel cytokines and chemokines to test the serum samples at baseline (PreIT), during therapy and after completion of all infusions of HER2 BATs (PostIT). Th₁ and Th₂ cytokines (IL-2, GM-CSF, TNF-α, IL-6, IL-10), chemokines (IP-10, MIP-1α, MIP-1β, MIP-3β, IL-8, GROα, GROβ, RANTES, MCP-1 and Fractalkine) and ligands for co-stimulatory (CD40L), and growth and differentiation receptors (Flt3L) using a Luminex System. The Th₁ cytokines IL-2 and GM-CSF showed median fold change of 8.0 and 1.9, respectively, and significantly increased levels at postIT (IL-2, p<0.01; GM-CSF, p<0.005) compared to preIT levels. Interestingly, Flt3L (p<0.01) and CD40L (p<0.02) both increased significantly at postIT compared to preIT baseline. Both, Flt3L and CD40L treatment have shown to be immune stimulatory and anti-tumor effects in prostate cancer (Figure. 5A) [10]. The Th₂ cytokines, IL-6 and IL-10 levels increased from preIT baseline to postIT levels but were not significant. The chemokines IP-10, MIP-1α, MIP-1β and Fractalkine were all significant with increases of 2.7 (p<0.001), 5.3 (p<0.04), 1.9 (p<0.03), and 1.7 fold (p<0.001), respectively, at postIT timeline compared to preIT baseline. Although IFN-γ was not detectable, it is intriguing that IT induced the IFN-γ induced chemokine IP-10 and the T cell recruiting chemokines MIP-1α and MIP-1β (Figure 5B). Together these immune data show that there is activation of a systemic endogenous immune effect induced by infusions of HER2 BATs. Levels of TNF-α, MIP-3β, RANTES, MCP-1, GROβ and IL-8 did increase significantly from preIT baseline.

Figure 5.

A) Profile of serum cytokines and chemokines. Analysis of sequential serum samples after HER2 BATs infusions (IT) show significant increase in Th1 cytokine levels (IL-2, p<0.01; and GM-CSF p<0.005) at postIT compared to preIT serum levels. None of the Th₂ cytokines (IL-6 and Il-10) showed significantly increased levels at postIT compared to preIT levels. Two growth factors, Flt3L (p<0.01) and CD40L (p<0.02), showed significant increase at postIT compared preIT baseline. B) Chemokines CXCL10 (IP-10, p<0.001), MIP-1α (p<0.04), MIP-1β (p<0.03) and fractalkine (p<0.001) increased at postIT compared to PreIT baseline levels.

Pattern of Vβ Repertoire Post IT.

Evaluation of Vβ repertoire in PBMC from four patients (KCI-03, KCI-04, KCI-07 and KCI-08) at preIT, midIT and postIT suggests detectable shifts in the Vβ repertoire detected by flow cytometric analysis (Figures S1A and S1B). We asked whether expended CD3⁺ TCR clones persisted in the PBMC of patient were able to produce cytoplasmic IFN-γ during IT and after completion of IT without any stimulation. The predominant T cell receptor (TCR) Vβ clones expanded in vivo after BATs infusions in three out of four patients also showed enhanced cytoplasmic IFN-γ production in these clones (KCI-03: Vβ14, 20; KCI-04: Vβ5.1; KCI-07: Vβ3, 5.1, 18) without any stimulation in the CD3⁺ T cells after IT. Some Vβ clones marginally expanded at mid- and/or postIT include (KCI-03: Vβ8 and 16; KCI-04: Vβ8, 16, 20, 21.3; KCI-07: Vβ2, 13.2, 17; KCI-08: Vβ17, 20). In contrast, some TCR clones (Vβ9, Vβ13.6, Vβ18, and Vβ23) may have expanded during IT but did not persist after 1 week of completion of all infusions. Other Vβ clones were decreased after IT (Figures S1A and S1B).

Survival and Kinetics of HER2 BATs in the circulation. Phenotyping for cells bearing OKT3 (mouse IgG2a) and the mid IT before the 5th infusion and 1 week (Figure S2) after the last infusion showed low but clearly detectable low levels of circulating IgG2a cells above the isotype and treatment baseline control.

Pretreatment Biopsy Cellular Infiltrates.

Scoring of Cell Densities and Expression Intensities of Immunostained Sections.

Quantitative analysis of IHC stained slides for tumor infiltrating immune cells (TIIC) was performed on nine tumor biopsy sections. The TIIC were evaluated for overall impression at low microscopic magnification (40x) followed by a higher magnification (100x– 200x) fields for tumor margin, stroma and tumor center. Five color stained sections (CD4+ T cells [Teal], CD8+ T cells (Purple), CD20+ B cells [green], CD68+ macrophages [Yellow] and FoxP3+ Tregs [Brown]) were scored as 0 (0–49) for the extremely low numbers of specific immune cells, 1 (50–100 specific immune cells) for sparse, 2 (101–1000 specific immune cells) for moderately dense, 3 (1001–5000 specific immune cells) for dense 4 (5000–10,000 specific immune cells) and for very dense T cells (CD4/CD8/Tregs), B cells or macrophages is scored 5 (>10,000 specific immune cells). Similar scoring was done for the single stained sections for IL-10 (Brown) and IFN-γ (Brown] and IFN-γ). The percentage of positive T-cell subpopulations was 100% for CD8 (9 of 9 patients), 88.9% for CD4 and FoxP3 positive T cells (8 of 9 patients), 22.4% for CD4 (15 of 67 patients), 78% for CD19+ B cells and CD68+ macrophages (7 of 9 patients), data is shown in Table S2 and Figures. 6A and 6B. Interestingly, PFS showed significant correlation with infiltration of CD4+ T cells (r=0.68, p<0.04) and FoxP3+ (r=0.85, p<0.003) T cells (Fig. 6A, lower panel). In Figures S3A–C, representative IHC results for two patient specimens (KCI-04 and KCI-11) are presented showing moderate to high densities of CD4+/CD8+ T cells, CD19+ B cells and low to moderate densities of FoxP3+ and CD68+ macrophages. Diagnostic biopsies from 3/4 patients with prolonged survival ranging from 28–36 months show high infiltration of both CD4+ and CD8+ T cells.

Figure 6.

A) Shows immune cell subsets by Multiplex IHC (mIHC) in 9/12 diagnostic metastatic prostate cancer biopsies. FFPE sections of PC were stained by 5-plex panel for CD4, CD8, FOXP3, CD19, CD68 and single staining for IFN-γ and IL-10. Quantitation data is displayed in the upper panel expressed as counts in a defined tumor area, lower panel shows correlation of cell density (counts) with overall survival. Positive correlation was observed for the presence of CD4+ T cells with prolonged OS (r=0.68; p<0.04). Interestingly, presence of FoxP3+ cells also favor prolonged OS (r=85; p<0.003). B) Shows the CD4, CD8, FOXP3, CD19, CD68 and expression of IFN-γ and IL-10 for each patient biopsy.

DISCUSSION

Despite the availability of multiple therapies for advanced mCRPC, immunotherapy remains an attractive modality due to the potential of inducing complete responses and durable remissions. The first FDA-approved cellular therapy in mCRPC; sipuleucel T was modestly successful in low volume and asymptomatic, or minimally symptomatic patients, with a small OS benefit [1]. Further clinical efforts with GVAX and ProstaVac, although promising in phase II trials, did not demonstrate adequate efficacy in randomized studies [2, 25, 26]. This is the first complete report of a clinical trial combining a checkpoint inhibitor with targeted T cell therapy, to assess clinical efficacy. Multiple other cellular therapies utilizing prostate specific membrane antigens or prostate specific stem cell antigens are in early testing [2].

Trials using pembrolizumab alone [27] or ipilimumab in combination with radiation therapy [16, 17] had limited success. In the pembrolizumab alone trial, a response rate of 3–5% was noted with median survival ranging from 9.5 to 14.1 months. In a 2022 press release, the KEYLYNK 010 study which compared pembrolizumab and olaparib combination to abiraterone or enzalutamide, failed to show a benefit. The study was stopped early for futility per the independent data monitoring committee recommendations The results suggest that pembrolizumab provided an immune response but was not impactful enough to manifest itself in clinically significant results. Immune therapy combinations need to be explored to enhance the effect. The key rationale behind combination of pembrolizumab and cellular therapy, was that the former would amplify the in situ immunization effect on the patient’s endogenous immune system provided by HER2 BATs.

This phase II study confirms the safety and suggests clinical efficacy for the combination in mCRPC. Ten patients completed all infusions of HER2 BATs without any major toxicities and 4 patients completed the entire course of combination immunotherapy. Adding pembrolizumab did not increase toxicities seen with HER2 BATs infusions. Majority of the adverse events were grades 1–2 and consisted of infusion reactions or fever, chills, headaches, nausea and/or myalgias. These lasted for 24–48 hours and were easily managed with symptom relief therapy. The remarkable finding is that 5 patients met the primary endpoint (progression free for more than 6 months). In addition PSA levels declined greater than 25% from baseline in 6 patients. Despite the enrollment of a highly pretreated patient population, 38.4% of patients were free of progression at 6 months. Four patients sustained responses ranging from 7.5 to 11.5 months. The remarkable tolerability and observed efficacy makes this regimen worthy of future investigation.

Our earlier phase I study in 7 mCRPC patients showed that HER2 BATs were safe and well tolerated, with 2 minor and 1 partial response. There were no severe adverse events noted. Three of 7 patients had decreases in PSA levels and 1 of 7 patients had a PSA that declined >50% below baseline. The PSA level decreases of 6 of 13 (46%) patients in the current trial is consistent with the PSA level decreases of 3 of 7 (43%) patients in our earlier phase I. Together, 45% (9 of 20) of the patients demonstrated a PSA response.

The immune evaluation in our earlier phase I trial showed enhanced specific cytotoxicity and increased Th₁ cytokines. Twelve patients in the current study were extensively evaluated for pharmacodynamic effects sequentially before, during and after combination immunotherapy.

Biomarker evaluations of the patients in this study showed a) significantly increased specific cytotoxicity against prostate specific cell line (PC3) after completion of BATs infusions (postIT) compared to preIT baseline, b) significantly decreased T regulatory cells postIT compared to baseline preIT time point, c) significantly increased levels of Th₁ cytokines IL-2 and GM-CSF after BATs infusions compared to baseline levels, d) Th₁ chemokines levels of IFN-γ induced IP-10 (CXCL10), MIP-1α, MIP-1β, and fractalkine were significantly elevated after immunotherapy (postIT). Although levels of Th₂ cytokines IL-6 and IL-10 increased in some patients, the difference was not statistically significant, e) The IFNγ responses in expanded Vβ clones strongly suggest endogenous specific TCR clonal response and development of CTL activation during therapy may be directed at multiple tumor antigens, and f) Although the numbers were not high, there were clearly detectable IgG2a+ HER2 BATs circulating in several patients. Together these immune data show that there is activation of a systemic endogenous immune effect induced by infusions of HER2 BATs. The study demonstrates that targeted cellular therapy approach holds promise and warrants further evaluation in mCRPC. As androgen receptor axis targeted therapies and chemotherapy are moved earlier, immunotherapy will likely fit in the space of frontline metastatic castrate resistant disease. There is an unmet need for immunologic biomarkers that can predict clinical response outcomes to enable selection of mCRPC cases likely to derive clinical benefit.

In conclusion, safety and promising efficacy was demonstrated with the combination of pembrolizumab and HER2BATs, and future investigation is warranted.

Translational Relevance.

The results of this phase II trial demonstrate that the combination of immune checkpoint inhibitor and patient specific armed activated T cells, is well tolerated and shows efficacy in prostate cancer. The toxicities consisted mainly of grade 1– 2 infusion reactions with fever, chills, headaches, nausea and/or myalgias. Six patients demonstrated PSA decline of 25% or greater. Five patients (38.4%) were free of progression at 6 months. The correlative studies demonstrate the proof of principle of immune stimulation in tumor tissue and in T cell subpopulations. This is the first study to demonstrate tolerability and efficacy of BATs in combination with immune checkpoint inhibition and further clinical investigation of the combination is warranted.