ABSTRACT
KRAS mutation hinders the therapeutic efficacy of epidermal-growth-factor-receptor (EGFR) mAb (cetuximab and panitumumab)-based immunotherapy of EGFR+ cancers. Although, cetuximab controls KRAS-mutated cancer cell growth in vitro utilizing a NK cell-mediated antibody-dependent-cellular-cytotoxicity-(ADCC) mechanism, KRAS-mutated colorectal carcinoma (CRC) cells can still escape NK cell immunosurveillance. To overcome this limitation, we used cetuximab and panitumumab to redirect Fcγ chimeric receptor (CR) T cells against KRAS-mutated HCT116 CRC cells. We compared 4 polymorphic Fcγ-CR constructs including CD16158F-CR, CD16158V-CR, CD32131H-CR, and CD32131R-CR which were transduced into T cells utilizing retroviral transduction. Percentages of transduced T cells expressing CD32131H-CR (83.5±9.5) and CD32131R–CR (77.7.±13.2) were significantly higher than those expressing with CD16158F-CR (30.3±10.2) and CD16158V-CR (51.7±13.7) (p<0.003). CD32131R-CR T cells specifically bound soluble cetuximab and panitumumab. However, only CD16158V-CR T cells released significantly higher levels of interferon gamma (IFNγ=1145.5 pg/ml ±16.5 pg/ml, p<0.001) and tumor necrosis factor alpha (TNFα=614 pg/ml ± 21 pg/ml, p<0.001) than non-transduced T cells when incubated with KRAS-mutated HCT116 cells opsonized with cetuximab. Only CD16158V-CR T cells combined with cetuximab controlled the growth of HCT116 cells subcutaneously engrafted in CB17-SCID mice. These results suggest that CD16158V-CR T cells combined with cetuximab represent useful reagents to develop an effective immunotherapy of EGFR+KRAS-mutated cancer.
INTRODUCTION
EGFR is overexpressed in several solid tumors 1. Upon binding to epidermal growth factor (EGF), EGFR triggers a series of signaling pathways supporting invasion and metastasis, and metastasis 1. The important role of EGFR in promoting cancer progression has provided the rationale to develop EGFR targeted mAb-based treatments 2.
EGFR-specific cetuximab is a chimeric IgG1 mAb preventing EGFR dimerization by stimulating its internalization and degradation. EGFR-specific panitumumab is a human IgG2 mAb interfering with EGF binding to its receptor. Cancer cells incubated with cetuximab or panitumumab undergo cell cycle arrest and apoptosis 2. However, a variety of EGFR+ cancer cells, including CRC cells, are insensitive to EGFR-specific mAbs since they carry RAS gene mutation(s) downstream of EGFR. Because of these mutations, cancer cells can bypass antitumor activities of both cetuximab and panitumumab. Lack of sensitivity of KRAS-mutated CRC cells to EGFR-specific mAb has serious clinical consequences since both cetuximab and panitumumab either have no effect on tumor growth or worsen CRC clinical course 3,4.
Increasing evidence suggests that this limitation can be overcome, at least for cetuximab, by taking advantage of its ability to mediate ADCC since EGFR+ cells, opsonized with cetuximab, undergo ADCC by activating the CD16 receptor expressed on NK cells 5. Nevertheless, cancer progression in patients is not arrested.
Failure to control cancer growth may be due to activation of evasion mechanism(s) from immune cells. However, in patients with cancer, NK cells, the major ADCC effector cells, show distinct functional defects and low ability to infiltrate solid tumors 6–8.
To restore the sensitivity of KRAS-mutated cancer cells to EGFR-specific mAbs, we investigated different strategies based on generation of extracellular CD16-CR linked to intracellular signaling and activating molecules 9–12. Because of NK cell limitations 13, we decided to use T cells, as effectors, since they easily infiltrate the tumor microenvironment and effectively protect hosts from cancer progression 14. Selected CD16-CR constructs have been transduced into T cells to redirect them by EGFR-specific mAbs toward EGFR+ cancer cells.
Other than NK cells, myeloid cells also mediate effector functions including proinflammatory cytokine production 15,16 and cytotoxic activity, including ADCC 15,17. Unlike NK cells, myeloid cells have the exclusive property to recognize Fc fragments of IgG2 antibodies complexed with the corresponding antigens on target cells, utilizing the Fc receptor CD32 and triggering ADCC activation 18. Both CD16 and CD32 are polymorphic and their polymorphisms influence their binding to IgG Fc fragments 19.
Still unknown is whether CD32 and CD16 polymorphisms impact the anti-tumor activity of Fcγ-CR T cells against KRAS-mutated CRC cells. The goal of this study is to compare the ability of polymorphic CD16-CR and CD32-CR to inhibit KRAS-mutated CRC cell proliferation and tumor progression in vitro and in vivo.
MATERIALS AND METHODS
Antibodies, reagents and cell lines
Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD3 (cat.555332), phycoerythrin (PE)-conjugated mouse anti-CD16 (cat.555407), PE-conjugated mouse antihuman CD32 (cat. 550586), mouse anti-human CD3 (cat. 555329), CD28 (cat.555725), CD32 (8.26) (cat. 557333), and CD16 (3g8) (cat. 556617) were purchased from BD Bioscience (San Jose, CA, USA). Cetuximab (Erbitux 5mg/ml) and panitumumab (Vectibix 20mg/ml) were purchased from Merck Serono (Darmstadt, Germany) and from Amgen (Thousand Oaks, CA, USA), respectively. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) was purchased from Sigma-Aldrich (Saint Louis, MO, USA) and GeneJuice® Transfection Reagent (Novagen) from Millipore (Burlington, MA, USA). Human recombinant interleukin-7 (IL-7) and interleukin-15 (IL-15) were purchased from Peprotech (London, UK) and Lipofectamine 2000 from Life Technologies (Carlsbad, CA, USA). Retronectin (Recombinant Human Fibronectin) was purchased from Takara Bio (Saint-Germain-en-Laye, France). Dulbecco’s Modified Eagle’s Medium (DMEM), Iscove’s Modified Dulbecco’s Medium (IMDM), RPMI 1640 medium, FBS, L-glutamine and penicillin/streptomycin were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Complete media (CM) were supplemented with 10% FBS, 2 mM L-glutamine, 0.1 mg/mL streptomycin, and 100U/ml penicillin. Mycoplasma-free, KRAS-mutated HCT116 cells 20, provided by Giulio Spagnoli (University of Basel, Basel, CH) were maintained in RPMI 1640, CM. Cells were authenticated on November 21th, 2018 by PCR-single-locus-technology (Eurofins, Ebersberg, Germany). Cells were kept in culture for a maximum of 4 to 8 passages.
Fcγ chimeric receptors
Generation of CD16158F-CD8α-CD28-CD3ζ CR has previously been described 12. Extracellular region of CD32A131R was amplified by reverse-transcriptase polymerase chain reaction (RT-PCR) from RNA extracted from peripheral blood mononuclear cells (PBMCs) utilizing the following primers: forward 5’-GAGAATTCACCATGACTATGGAGACCCAAATG-3’ and reverse 5’-CGTACGCCCCATTGGTGAAGAGCTGCC-3’ (Thermo Fisher Scientific, Waltham, MA, USA). PCR product was fused by restriction enzyme-compatible ends with the CD8α-CD28-CD3ζ domain contained in the pcDNA3.1/V5-His TOPO TA (Invitrogen, Carlsbad, CA, USA). CD16158F-CR and CD32131R-CR were subcloned into NcoI and MluI sites of the SFG retroviral vector. CD16158V and CD32A131H were assembled by using synthetic oligonucleotides. Fragments were separately inserted into SFG vector and sequenced by GeneArt Gene Synthesis team (Invitrogen-Thermo Fisher Scientific, Regensburg, Germany).
Retrovirus production and T cell transduction
Retroviral supernatants were obtained by transient transfection of 293T cells, with Peg-Pam plasmid encoding the Moloney murine leukemia virus gag and pol genes, and RDF plasmid encoding the RD114 envelope and the CD32131R-, CD32131H-, CD16158F- or CD16158V-CR SFG retroviral vectors, using GeneJuice reagent. Forty-eight and 72h posttransfection, retrovirus-containing supernatants were harvested, filtered, snap frozen, and stored at −80°C until use. To generate Fcγ-CR T cells, PBMCs (0.5×106 PBMCs/ml) were cultured for 3 days in non-tissue culture treated 24-well plates pre-coated with 1 μg/ml anti-CD3 and 1 μg/ml anti-CD28 mAbs in the presence of 10 ng/ml IL-7 and 5 ng/ml IL-15. Viral supernatants were placed on retronectin-coated non-tissue culture treated 24 well plates and spun for 1.5h at 2000xg. Activated T cells were seeded into retrovirus loaded-plates, spun for 10’, and incubated for 72h at 37°C in 5% CO2 atmosphere. After transduction, T cells were expanded in RPMI 1640 CM supplemented with 10 ng/ml IL-7 and 5 ng/ml IL-15.
In vitro tumor cell viability assays
Antitumor activity of Fcγ-CR T cells in vitro was evaluated by MTT assays. Tumor target cells (7×103/well) were seeded in 96-well plates and Fcγ-CR T cells (35×103/well) were added in the presence or absence of (3 μg/ml) cetuximab or panitumumab. Following a 48-72h incubation at 37°C, non-adherent T cells were removed and 100 μl/well of fresh medium supplemented with 20 μl of MTT (5 mg/ml) were added to adherent cells and incubation was continued for 3h at 37°C. Following a 3h incubation at 37°C, supernatants were removed and 100 μl of dimethyl sulfoxide (DMSO) were added to each well. Absorbance was measured at 570 nm.
Cytokine release
Two-fold dilutions of a Fcγ-CR T cell suspension (1×105/100μl/well) were added to 96 well plates in triplicates. Then, EGFR+, KRAS-mutated HCT116 cells (2×104/100μl/well) were added at a 5:1 E: T ratio in the presence or absence of 3μg/ml cetuximab or panitumumab. Supernatants were collected following a 48h incubation at 37°C and IFNγ and TNFα levels were measured by ELISA (Thermo Fisher Scientific, Waltham, MA, USA).
Flow cytometry
Fcγ-CR expression levels on engineered T cells were assessed by flow-cytometry upon incubation for 30min at 4°C with FITC-conjugated mouse anti-human CD3, PE-conjugated mouse anti-human CD32 or PE-conjugated mouse anti-human CD16 mAbs. Cells were analyzed by 2-laser BD FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA) flow cytometer. Results were evaluated utilizing Tree Star Inc. FlowJo software.
Xenograft mouse model
In vivo experiments were performed in accordance with the directive 2010/63/EU and authorized by the Italian Ministry of Health (186/2016-PR). Antitumor activity of CD16158V-CR T cells with or without cetuximab was assessed using 8-week-old male CB17-SCID mice (CB17/lcr-PrkdcSCID/lcrlcoCrl, Charles River Laboratories, Lecco, Italy, Cat.CRL:236, RRID: IMSR CRL:236), 12-18gr body weight, engrafted with KRAS-mutated HCT116 CRC cells. Mice were housed in temperature-controlled rooms with 12h light/dark cycle and free access to sterile water and autoclaved standard chow diet (4RF25; Mucedola, Milan, Italy). Endogenous NK cell activity was suppressed by intraperitoneal injection of 20μl rabbit anti-asialo-GM1 antibody (Wako, Chemicals, Richmond, VA, Cat.986-10001). Mice received anti-asialo-GM1 antibody on days −3, 0, +14, and +21 since tumor cell engraftment. On day 0, mice were grafted subcutaneously in the right flank, with 1×106 HCT116 cells and then randomly separated into 4 groups (5 mice per group). Group 1 received only HCT116; group 2 cetuximab (150μg); group 3 HCT116 and CD16158V-CR T cells and group 4 HCT116, CD16158V-CR T cells and cetuximab (150 μg). Effector cells were administered at a 5:1 E:T ratio. Tumor volumes were measured every 3 days with caliper and calculated using the formula: TV (cm3) = 4/3p r3, where r= (length + width)/4. When tumor volume reached 2 cm3 mice were sacrificed.
Statistical analysis
Results were analyzed by paired-T-test, Mann-Whitney test and two-way analysis of variance (ANOVA) followed by Bonferroni’s multiple-comparison, as necessary. Disease-free survival (DFS) was evaluated by log-rank-(Mantel-Cox) test. Differences were considered significant with p-values < 0.05.
RESULTS AND DISCUSSION
To enhance anti-tumor potential of EGFR-specific mAbs, we generated CD16 and CD32-CR (fig.1A). We successfully expressed all indicated Fcγ-CR into transduced T cells (fig.1B). However, T cell transduction of CD32131H-CR (83.5%±9.5%) and CD32131R-CR (77.7%±13.2%) was significantly more effective than that of CD16158V-CR (51.7%±13.7%) and CD16158F-CR (30.3%±10.2%) (p<0.003 fig.1C). Taking in consideration that all the retroviruses are packaged using the same methodology and that the titer of the produced viruses are similar, these results may suggest that in human T lymphocytes polymorphic CD32-CR are more stably expressed as compared to polymorphic CD16-CR even if the underlying mechanism(s) remain to be explored. However, Cheeseman et al., provided evidence that hematopoietic cells tend to express CD32 more efficiently than CD16 on their surfaces 21.
To evaluate Fcγ-CR T cell antibody binding capacity, polymorphic CD32-CR and CD16-CR T cells were incubated with cetuximab or panitumumab, for 30min, at 4°C. CD32131R-CR T cells efficiently bound both cetuximab and panitumumab while CD32131H-CR T cells only displayed a minimal binding for panitumumab. In contrast, CD16158V-CR T cells and CD16158F-CR T cells failed to bind both cetuximab and panitumumab (fig.1D). Binding of cetuximab and panitumumab to CD32-CR T cells was highly specific and promptly inhibited in the presence of Fc receptor blocking reagent (FcR BR) (fig.1D). CD16 and CD32 binding affinity for IgG is known to be influenced by their polymorphisms. Presence of valine instead of phenylalanine at position 158 of CD16 (CD16158V/V) and presence of histidine instead of arginine at position 131 of CD32 (CD32131H/H) enhance IgG binding affinity of these receptors 19,22. CD16-CR has also been produced in other laboratories while, to the best of our knowledge, CD32-CR has not. CD16158V has preferentially been utilized for in vitro and in vivo studies 9–11,23. However, Kudo et al. showed that CD16158V-CR has significantly higher affinity for rituximab than CD16158F-CR. Here, neither CD16158V-CR nor CD16158F-CR showed significant binding affinity for soluble cetuximab and panitumumab. Inability of our CD16-CR to bind cetuximab, might be related to structural differences of endodomains since we used CD28 while Kudo et al. used 4-1BB. CD32131R-CR bound soluble cetuximab and panitumumab with higher affinity than CD32131H-CR. As observed with CD16-CR, structural differences of CD32-CR endodomains may also influence their IgG binding affinity.
We next asked whether polymorphic Fcγ-CR T cells recognize KRAS-mutated HCT116 cells opsonized with EGFR-specific mAb and affect their viability. Transduced and non-transduced T cells were incubated, for 72h, at 37°C with KRAS-mutated HCT116 cells with or without cetuximab or panitumumab. IFNγ and TNFα production was measured in culture supernatants. Only CD16158V-CR T cells, combined with cetuximab but not panitumumab, produced levels of both IFNγ (1145.5±16.5 pg/ml) and TNFα (614±21 pg/ml) significantly higher than those released by the other Fcγ-CR T cells or nontransduced T cells (fig.2A).
Then, we tested whether polymorphic Fcγ-CR engineered T cells could impair viability of KRAS-mutated HCT116 cell opsonized with cetuximab or panitumumab, utilizing an ADCC mechanism. Figure 2B shows that KRAS-mutated HCT116 cell viability, expressed in optical density (OD), was significantly reduced following a 48h incubation at 37°C with CD16158V-CR T cells and cetuximab (0.67-OD±0.03-OD) as compared to nontransduced T cells (1.5-OD±0.03-OD). In contrast, no change in viability was detected when cetuximab was replaced with panitumumab. Both polymorphic CD32-CR T cells and CD16158F-CR T cells with or without anti-EGFR mAbs, failed to impair KRAS-mutated HCT116 cell viability. These data indicate that the efficient recognition of cetuximab-opsonized cancer cells by CD16158V-CR T cells leads to the activation of effector functions including production of proinflammatory cytokines and impairment of KRAS-mutated HCT116 cell viability. Therefore, CD16158V-CR T cells restore the ability of cetuximab to target KRAS-mutated HCT116 CRC cells by an immune-mediated mechanism in vitro.
Since these data challenged the effector potential of CD16158F-CR T cells and CD32-CR T cells, we tested them on KRAS-mutated HCT116 cells stably transfected with CD32 (Caratelli et al. unpublished data) in redirected ADCC assays. Although to different extents, all CR-engineered T cells, at different E: T ratio, significantly reduced the viability of FcγR+HCT116 cells in the presence of 3g8 (anti-CD16) or 8.26 (anti-CD32) agonistic mAb (Figure 2C). These data indicate that all engineered T cells are fully competent effector cells. Thus, although transgenic CD32 binds soluble mAbs and is able to provide a cytotoxic signal, CD32-CR T cells are unable to produce pro-inflammatory cytokines in coculture with opsonized KRAS-mutated HCT116, and to impair their viability. Therefore, soluble mAb binding and redirected killing do not represent effective surrogate assays for the ability of transduced T cells to elicit mAb-mediated effector functions targeting tumor cells. Notably, affinity of IgG1 cetuximab for CD32 is low and panitumumab mediates ADCC only in the presence of myeloid cells 18.
In vivo antitumor potential of CD16158V-CR T cells was then assessed. Transduced cell ability to impair KRAS-mutated HCT116 viability was tested prior to their administration to experimental animals. CD16158V-CR T cells were incubated at 37°C with target cells and cetuximab or panitumumab while non-transduced T cells were used as a negative control. Following 72h incubation, KRAS-mutated HCT116 viability was assessed by the MTT assay. Figure 3A confirms that co-culture with CD16158V-CR T cells, combined with cetuximab, significantly affected HCT116 cell viability. Instead, CD16158V-CR T alone or with panitumumab or non-transduced T cells were completely ineffective. We then engrafted CB17-SCID mice subcutaneously with KRAS-mutated HCT116 cells. One hour later CD16158V-CR T cells, with or without cetuximab, were injected in proximity of the injection site of HCT116 cells. Figure 3B shows tumor volumes of HCT116 on day 64 postinjection. CD16158V-CR T cells combined with cetuximab, significantly protected mice from tumor growth. DSF of treated animals is reported in figure 3C. Interestingly, tumor growth was also significantly delayed in the group of mice receiving CD16158V-CR. These results indicate that CD16158V-CR T cells combined with cetuximab and, to a lesser extent, even without cetuximab, exert significant anti-tumor activity in vivo.
This is the first study demonstrating the ability of CD16158V-CR combined with cetuximab can control KRAS-mutated cancer cell growth in vitro and in vivo. Our results by using the same KRAS-mutated HCT116 cells and at an equivalent reverse ADCC potential of Fcγ-CR utilized, the superior activity of CD16158V-CR may reflect its optimal interaction with the cetuximab Fc fragment 5. Taken together, these data contribute to a repositioning of currently available anti-EGFR therapeutic mAbs in the treatment of insensitive tumors, and pave the way toward innovative immunotherapies targeting KRAS mutated cancers.
ACKNOWLEDGMENTS
This work was supported by the Italian Association for Cancer Research (AIRC) under grant IG17120 to GS and by NIH grants CA216114 and CA231766 to SF. We thank Marta Coccia and Antonio Rossi for technical assistance
Footnotes
Disclosure of Potential Conflicts of Interest: The authors declare no potential conflict of interest.
Novelty and Impact: The major limitation of the monoclonal antibody(mAb)-targeted therapy of epidermal growth factor receptor (EGFR)+ colorectal carcinoma (CRC) is(are) KRAS mutation(s) downstream EGFR. Here, we demonstrate that this limitation can be overcome by redirecting, in vitro and in vivo, T cells engineered with CD16158V-chimeric receptor by cetuximab. This study may help develop efficient immunotherapy of EGFR+KRAS-mutated CRC.
- Abbreviations
- EGF
- epidermal growth factor
- EGFR
- epidermal-growth-factor-receptor
- mAb
- monoclonal antibody
- CRC
- colorectal carcinoma
- ADCC
- antibody-dependent-cellular-cytotoxicity
- CR
- chimeric receptor
- INFγ
- interferon gamma
- TNFα
- Tumor necrosis factor alpha
- FITC
- fluorescein isothiocyanate
- IL-7
- interleukin-7
- IL-15
- interleukin-15
- RT-PCR
- reverse-transcriptase polymerase chain reaction
- PBMCs
- peripheral blood mononuclear cells
- DMEM
- Dulbecco’s Modified Eagle’s Medium
- IMDM;
- Iscove’s Modified Dulbecco’s Medium
- CM
- complete media
- DMSO
- dimethyl sulfoxide
- DSF
- disease-free survival
- FcR BR
- Fc receptor blocking reagent
- OD
- optical density