ABSTRACT
Cetuximab and panitumumab bind the human epidermal growth factor receptor (EGFR). While the chimeric cetuximab (IgG1) triggers antibody-dependent-cellular-cytotoxicity (ADCC) of EGFR positive target cells, panitumumab (a human IgG2) does not. The inability of panitumumab to trigger ADCC reflects a poor binding affinity of human IgG2 Fc for the FcγRIII (CD16) on NK cells. However, both human IgG1 and IgG2 bind the FcγRII (CD32) to a similar extent. Here, we have compared the ability of T cells, engineered with a novel low-affinity CD32131R -chimeric receptor (CR), and those engineered with the low-affinity CD16158F–CR T cells in eliminating EGFR positive epithelial cancer cells (ECCs) in combination with cetuximab or panitumumab. Following T cell transduction, the percentage of CD32131R-CR T cells was (74±10) significantly higher than that of CD16158F-CR T cells (46±15). Only CD32131R-CR T cells bound panitumumab. CD32131R-CR T cells combined with the mAb 8.26 (anti-CD32) and CD16158F-CR T cells combined with the mAb 3g8 (anti-CD16) eliminated colorectal carcinoma (CRC), HCT116FcγR+ cells, in a reverse ADCC assay in vitro. Cross-linking of CD32131R-CR on T cells by cetuximab or panitumumab and CD16158F-CR T cells by cetuximab induced elimination of triple negative breast cancer (TNBC) MDA-MB-468 cells, and secretion of IFN gamma (IFNγ) and tumor necrosis factor alpha (TNFα). Neither cetuximab nor panitumumab induced Fcγ-CR T anti-tumor activity against KRAS-mutated HCT116, non-small-cell-lung-cancer, A549 and TNBC, MDA-MB-231 cells. ADCC of Fcγ-CR T cells was significantly associated with the over-expression of EGFR on ECCs. In conclusion, CD32131R-CR T cells are efficiently redirected by cetuximab or panitumumab against BC cells overexpressing EGFR.
Article category Tumor Immunology and Microenvironment
Novelty and Impact Monoclonal antibody-redirected Fcγ-CR T cell immunotherapy represents a promising approach in the fight against cancer. Here, we expand the application of this methodology to TNBC overexpressing the EGFR utilizing a novel CD32A131R-CR in combination with anti-EGFR mAbs. Our study supports the use of CD32A131R-CR T cells combined with panitumumab or cetuximab for targeting TNBC cells overexpressing the EGFR. Our results may be utilized as a platform for the rational design of therapies targeting TNBC overexpressing EGFR.
INTRODUCTION
The lytic activity of ADCC is influenced by multiple variables including the type of affinity by which the Fc fragment of an antibody binds to the FcγR on a competent cytotoxic cell, the expression level of the targeted antigen on the surface of targeted cells, and the association constant of the antibody for the surface antigen of interest 1. The FcγR family includes CD16, CD32, and CD64. The former two receptors can be expressed in polymorphic forms, each of which displays different binding affinity for the Fc portion of IgG. The presence of valine at position 158 of CD16 (CD16158V) and of histidine at position 131 of CD32 (CD32131H) identifies the high-affinity receptors while the presence of phenylalanine at position 158 (CD16158F) and of arginine at position 131 (CD32131R) of CD16 and CD32, respectively, defines low-affinity receptors 2. Also, the activity of ADCC is influenced by the mAb subclasses since distinct subclasses have different association constants for the FcγRs CD16 and CD32. Cetuximab (IgG1) and panitumumab (IgG2) are currently utilized for the treatment of EGFR positive tumors. These two mAbs have demonstrated differential ability to mediate cell dependent cytotoxicity against EGFR positive epithelial cancer cells (ECCs) 3. Only cetuximab mediates CD16 positive NK cell-dependent cytotoxicity of EGFR positive cancer cells. The differential activity of cetuximab and panitumumab reflects the low affinity of IgG2 for the FcγR CD16 4. In contrast, CD32 binds both IgG1 and IgG2 although with different affinity 2.
The role of ADCC in the in vitro and in vivo antitumor activity of tumor antigen (TA)-specific mAbs 5 has stimulated interest in genetically engineering T cells with the CD16 chimeric receptor (CD16-CR) 6. In these cells, the extracellular domain of CD16 was ligated to cytotoxic signaling molecules fused with 7,8 or without 9,10 T cell costimulatory molecules. This strategy allows the rapid generation of polyclonal T cells with a potent cytotoxic activity when combined with mAbs recognizing TAs expressed on tumor cell membrane. Based on this background information, we have utilized cetuximab and panitumumab as a model to demonstrate that CD32131R-CR T cells have higher cytotoxic activity than CD16158F-CR T cells since they eliminate EGFR positive cancer cells in combination with both cetuximab and panitumumab. In the present study, we have engineered T cells with a novel second generation of CD32131R-CR. The anti-tumor activity of CD32131R-CR T cells was compared to that of CD16158F-CR T cells in combination with cetuximab (IgG1) or panitumumab. Both engineered T cells, in combination with cetuximab, exerted a significant anti-tumor activity against breast cancer (BC) cells overexpressing the EGFR (EGFRhigh). However, only CD32131R-CR T cells effected cytotoxicity against EGFRhigh BC cells in combination with either cetuximab or panitumumab. Our results strongly suggest that CD32131R-CR has a potential to be utilized in Fcγ-CR T cell-based immunotherapy of EGFR overexpressing BC cells.
MATERIALS AND METHODS
Antibodies and Reagents
Allophycocyanin (APC)-conjugated anti-human CD3 (cat. 555335), fluorescein isothiocyanate (FITC)-conjugated anti-human CD3 (cat. 555332), FITC-conjugated anti-human CD107A (cat. 555800), phycoerythrin (PE)-conjugated anti-human CD16 (cat. 555407), PE-conjugated anti-human CD32 (cat. 550586), FITC-conjugated mouse anti-human IgG (cat. 555786), FITC-conjugated goat anti-mouse IgG (cat. 555748), mouse anti-human CD3 (cat. 555329), and anti-human CD28 (cat. 555725) were purchased from BD Bioscience (San Jose, CA, USA). Mouse anti-human CD16 (clone 3g8), mouse anti-human CD247 (CD3ζ) mAb (clone 6B10.2) and mouse anti-human EGFR antibody (clone AY13) were purchased from Biolegend (San Diego, CA, USA). Anti-human CD32 mAb (clone 8.26) was purchased from BD Bioscience (San Diego, CA). Anti-human B7-H3 (CD276) mAb 376.96 was developed and characterized as described 11. mAb 376.96 was purified from ascitic fluid by affinity chromatography on Protein A. The activity and purity of mAb preparations was monitored by binding assays and SDS-PAGE. Cetuximab (Erbitux) and Panitumumab (Vectibix) were from Merck Serono (Darmstadt, Germany) and Amgen (Thousand Oaks, CA, USA), respectively. Anti-phospho Tyr142 (Y142) CD3ζ mAb (cat. ab68235) was purchased from Abcam (Cambridge, UK). 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) was obtained from Sigma-Aldrich (Saint Louis, MO, USA). FcR blocking reagent (BR) was purchased from Miltenyi (Bergisch Gladbach, Germany). GeneJuice® Transfection Reagent (Novagen) was from Millipore (Burlington, MA, USA). Human recombinant interleukin-7 (IL-7) and interleukin-15 (IL-15) were from Peprotech (London, UK). Lipofectamine 2000 was from Life Technologies (Carlsbad, CA, USA), and Retronectin (Recombinant Human Fibronectin) was purchased from Takara Bio (Saint-Germain-en-Laye, France).
Cell lines
The 293T packaging cell line was used to generate the helper-free retroviruses for T cell transduction. 293T cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% Fetal Bovine Serum (FBS), 2mM L-glutamine, 0.1mg/mL streptomycin and 100U/ml penicillin hereafter referred to as IMDM complete medium (CM). KRAS-mutated A549 and HCT116 cell lines were maintained in RPMI-1640 CM. KRAS-mutated TNBC cells, MDA-MB-231, and KRAS wild-type TNBC cells, MDA-MB-468, were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) CM (Thermo Fisher Scientific, Waltham, MA, USA). 293T cells were kindly provided by Dr. Gianpietro Dotti, University of North Carolina, Chapell Hill, USA. A549 cells were kindly provided by Dr. Antonio Rossi, National Research Council, Italy. MDA-MB-231 and MDA-MB-468 cells were kindly provided by Dr. Maria Lucibello, National Research Council, Italy. HCT116 cells were kindly provided by Dr. Giulio Cesare Spagnoli, University of Basel, Switzerland. Mycoplasma-free cancer cell lines utilized in our study are part of our lab collection. Authentication test was successfully performed on November 21th, 2018 by PCR-single-locus-technology (Eurofins, Ebersberg, Germany). Cell lines were passaged for 4 to 8 times before use or kept in culture for a maximum of 6 weeks.
CD32131R-CR construction
The signal peptide (nucleotide 1 - 101) and the extracellular region (nucleotide 102 - 651) of the low-affinity variant CD32A131R, hereafter referred to as CD32131R, was amplified by reverse-transcriptase polymerase chain reaction (RT-PCR) from RNA extracted from freshly isolated peripheral blood mononuclear cells (PBMCs) utilizing the following primers: forward 5’-GAGAATTCACCATGACTATGGAGACCCAAATG-3’ and reverse 5’-CGTACGCCCCATTGGTGAAGAGCTGCC-3’ (Thermo Fisher Scientific, Waltham, MA, USA). The PCR product was fused in tandem by restriction enzyme-compatible ends with the CD8α transmembrane domain and the CD28 and CD3ζ intracellular regions already available in the lab (CD32131R-CR). The generation of CD16158F-CR has already been described 8. The CD32131R-CR and CD16158F-CR genes were subcloned into the NcoI and MluI sites of the SFG retroviral vector.
Retrovirus production and T cell transduction
Retroviral supernatants were obtained by transient transfection of 293T packaging cells, using the GeneJuice reagent, with the following vectors: the Peg-Pam vector containing the Moloney murine leukemia virus gag and pol genes, the RDF vector containing the RD114 envelope and the CD32131R-CR or CD16158F-CR SFG retroviral vectors. Forty-eight and 72h post-transfection, the conditioned medium containing the retrovirus was harvested, filtered, snap frozen, and stored at −80°C until use. For the generation of Fcγ-CR T cells, PBMCs (0.5×106 PBMCs/ml) were cultured for 3 days in a non-tissue culture treated 24-well plate pre-coated with 1μg/ml of anti-CD3 and 1μg/ml of anti-CD28 mAbs in the presence of 10 ng/ml of IL-7 and 5ng/ml of IL-15. The viral supernatant was loaded on retronectin-coated non-tissue culture treated 24 well plates and spun for 1.5h at 2000xg. Activated T cells were seeded into the retrovirus loaded-plate, spun for 10’, and incubated for 72h at 37°C in 5% CO2. After transduction, T cells were expanded in RPMI-1640 CM supplemented with 10ng/ml of IL-7 and 5 ng/ml of IL-15 for 12-13 days and analyzed.
Western blot
CD32131R-CR transduced and non-transduced T cells were lysed with Triton buffer composed of 1% (v/v) Triton X-100, 20mM Tris-HCL pH7.6, 137mM NaCl, 1mM MgCl2, 1mM CaCl2, 2mM phenylmethylsulfonyl fluoride (PMSF) supplemented with phosphatase (Sigma-Aldrich, Saint Louis, MO, USA) and protease (Roche, Basel, Switzerland) inhibitor cocktails. Thirty micrograms of protein lysates were resolved on Bolt 4-12% Bis-Tris plus gel (Invitrogen, Carlsbad, CA, USA) under reducing conditions and transferred to a nitrocellulose filter. The filter was probed overnight at 4°C with a mouse anti-human CD3ζ or rabbit anti-phospho-tyrosine CD3ζ (Y142) antibody. The latter was detected utilizing a horseradish peroxidase-conjugated donkey anti-mouse (Jackson Laboratory, Bar Harbor, ME, USA) for 1h at room temperature. Antibody binding was visualized with Amersham ECL Western blotting detection reagent (GE Healthcare, Little Chalfont, UK).
Binding assay
A direct immunofluorescence analysis was utilized to test the Fc antibody-binding ability of a FITC-conjugated anti-CD107A mAb to the CD32131R-CR and CD16158F-CR. CD32131R-CR and CD16158F-CR T cells were incubated with 5μl of FITC-conjugated anti-CD107A, with or without Fc receptor blocking reagent (FcR BR) for 30 min at at 4°C. Then, cells were washed and analyzed by flow cytometry. Cetuximab or panitumumab Fc fragment binding to CD32131R-CR or CD16158F-CR T cells was evaluated by staining with a FITC-conjugated anti-human IgG.
Flow cytometry
Expression of CD32131R-CR or CD16158F-CR on transduced T cells was assessed by staining for 30 min at 4°C with FITC-conjugated anti-human CD3, PE-conjugated anti-human CD32 or PE-conjugated anti-human CD16 mAbs, respectively. Cells were then analyzed by a 2-laser BD FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA) flow cytometer. Results were analyzed utilizing Tree Star Inc. FlowJo software.
Cytokine release assay
CD32131R-CR or CD16158F-CR transduced T cells (2×105/well) were added to 96 well plates previously coated with 10μg/ml of anti-CD3, 3g8 or 8.26 mAbs. In co-culture experiments, CD16158F-CR T cells or CD32131R-CR T cells were plated in 96-well plates with target cell lines at 5:1 E:T ratio in the presence or absence of 3μg/ml of cetuximab or panitumumab or the anti-B7-H3 mAb, 376.96. Supernatants were collected after 24 or 48h of culture. IFNγ and TNFα levels were measured by ELISA (Thermo Fisher Scientific, Waltham, MA, USA).
In vitro tumor cell viability assay
Tumor target cells (7×103/well) were seeded into 96-well plates and CD16158F-CR T cells or CD32131R-CR T cells (35×103/well) were added in the presence or absence cetuximab or panitumumab or the anti-B7-H3 mAb 376.96 (3μg/ml) (see above). Following a 48h incubation at 37°C, non-adherent T cells were removed. Then a suspension of fresh medium (100μl/well) supplemented with MTT (20μl/5mg/ml) was added to the adherent cells for 3h at 37°C. MTT was then removed and 100μl of dimethyl sulfoxide was added to each well. Absorbance (optical density, OD) was measured at 570 nm.
Statistical analysis
Results were analyzed by a Paired-T-test or a Mann-Whitney test. The relationship between the two variables was measured by the Spearman’s rank correlation coefficient. Differences with p-value < 0.05 were considered significant.
RESULTS
CD32131R and CD16158F CRs are differentially expressed on T cells
Activated T cells were transduced in vitro with a gamma-retroviral vector encoding the CD32131R-CR (fig. 1A). Cells were then tested for expression of CD32131R-CR by western blot and flow cytometry analysis. For biochemical analysis, we utilized two mAbs specific for the non-phosphorylated and phosphorylated CD3ζ chain. Both mAbs detected 2 distinct bands. The band of a MW slightly higher than 51 kDa matches with the expected size of the CD32131R-CR while the smaller 18 kDa band detected in both control and CD32131R-CR expressing T cells corresponds to the endogenous CD3ζ chain (fig. 1B). By flow cytometry, CD32131R-CR was clearly detectable on the cell surface of engineered T cells (fig. 1C, left panel). Transduction efficiency of CD32131R-CR was significantly higher than that of CD16158F-CR (74% ± 10% vs. 46% ± 15%, p<0.001) (fig. 1C, right panel).
CD32131R-CR specifically bound the Fc fragment of soluble immunoglobulins
In initial experiments, we compared the ability of CD32131R-CR and CD16158F-CR T cells to bind soluble IgG Fc fragment. As a model reagent, we chose the H4A3 mAb, a FITC-conjugated IgG1 specific for CD107A, an intracellular lysosomal-associated membrane protein (LAMP-1). We first evaluated the binding of anti-CD107A mAb on the surface of CD32131R-CR T cells in comparison to CD16158F-CR T cells. Following a 30 min incubation at room temperature, CD32131R-CR T cells effectively bound anti-CD107A mAb on their surfaces (fig. 2A, left panel) whereas CD16158F-CR did not (fig. 2A, right panel). Binding was highly specific since it was abrogated in the presence of FcR blocking reagent (BR). To further evaluate whether CD32131R-CR T cells were capable of binding mAb Fc fragment, in a more physiological condition, we tested whether the Fc fragment-binding capacity of CD32131R-CR was preserved in the presence of human immunoglobulins. Therefore, we incubated anti-CD107A with CD32131R-CR T cells in a buffer containing 10% of human plasma (fig. 2B). Following a 30 min incubation, at room temperature, anti-CD107A mAb was still bound to engineered T cells (fig. 2B).
To assess the Fcγ-CR T cell potential to target EGFR+ ECCs, upon incubation with anti-EGFR mAbs, we tested their antibody-binding capacity by utilizing cetuximab and panitumumab mAbs. Only CD32131R-CR T cells bound the Fc fragment of both soluble anti-EGFR mAbs (fig. 2C middle, panels) with higher binding capacity for cetuximab, as compared to panitumumab (fig. 2C, lower panels). Of note, binding of both mAbs was abolished in the presence of FcR BR (fig. 2C). Finally, we performed a dose-response binding assays, in which cetuximab was incubated at increasing concentrations with the Fcγ-CR T cells for 30 min at 4°C. As shown in Fig. 2D, only CD32131R-CR T cells (solid lines) bound cetuximab. The maximum binding capacity of CD32131R-CR, expressed as both percentages and MFI of positive cells, was achieved at concentrations ranging between 1-10 μg/ml of cetuximab. These results demonstrate that CD32131R-CR T cells have a superior binding ability for soluble mAbs than CD16158F-CR T cells.
CD32131R-CR and CD16158F-CR T cells eliminate KRAS-mutated HCT116FcγR+ cells in redirected ADCC assays and release IFNγ and TNFα upon specific antigen stimulation
Next, we tested the ability of Fcγ-CR-transduced T cells to elicit cytotoxic activity in a reverse ADCC assay (fig. 3A). To this end, CD32131R-CR T cells and CD16158F-CR T cells were incubated in the presence of HCT116 cells, stably transfected with CD32, in the presence of the anti-CD32 and anti-CD16 mAbs respectively. Tumor cell viability was analyzed after 48h by MTT assay. Incubation with either CD32131R-CR T cells or CD16158F-CR T cells significantly reduced numbers of viable HCT116FcγR+ cells, consistent with efficient Fc-mediated cytotoxic activity. Furthermore, cross-linking of either Fcγ-receptor with specific mAbs induced the release of comparable amounts of IFNγ and TNFα (fig. 3B). These data indicate that both CD32131R-CR T cells and CD16158F-CR T cells clearly mediate comparable levels of reverse ADCC when given in combination with the mAbs 8.26 and 3g8 respectively.
CD32131R-CR T cells and CD16158F-CR T cells differ in their ability to eliminate MD-MB-468 cells, in combination with cetuximab and panitumumab
The ability of CD32131R-CR to specifically bind the cetuximab Fc fragment prompted us to investigate whether this binding triggers ADCC against EGFR+ cancer cell lines. CD32131R-CR T cells and CD16158F-CR T cells were incubated with many ECC lines at an E:T ratio of 5:1. Tumor cell viability was assessed following a 48h incubation at 37°C. CD32131R-CR T cells significantly reduced the viability of MDA-MB-468 cells in the presence of cetuximab or panitumumab while CD16158F-CR T cells were only effective in the presence of cetuximab (fig. 4). In contrast, anti-B7-H3 379.96 mAb, which stained MDA-MB-468 cells, did not cause any detectable change in ECC viability. Neither cetuximab nor panitumumab had detrimental effects on MDA-MB-468 cells in the absence of Fcγ-CR T cells (fig. 4). However, CD32131R-CR T cells and CD16158F-CR T cells in combination with cetuximab or panitumumab failed to affect the viability of EGFR+ MDA-MB-231, A549, and HCT116 cells (fig. 4).
Crosslinking of CD16158F-CR on engineered T lymphocytes cultured with MDA-MB-468 breast cancer cells promoted the release of IFNγ (fig. 5A) and TNFα (fig. 5B) in the presence of cetuximab but not of panitumumab. In contrast, both mAbs triggered the release of both cytokines by CD32131R-CR engineered T cells.
Furthermore, although panitumumab failed to mediate cell dependent cytotoxicity against the A549 and HCT116 cell lines, it induced a significant release of IFNγ by CD32131R-CR T cells (fig.5A) incubated with the two cell lines.
Correlation of EGFR expression level on targeted cancer cell lines with the cetuximab dependent FcγCR T cell cytotoxicity
Our results indicate that among the EGFR+ cancer cell lines evaluated, only MDA-MB-468 cells were efficiently killed by CD16158F-CR T cells in combination with cetuximab and by CD32131R-CR T cells in combination with cetuximab or panitumumab (fig. 4). Since MDA-MB-468 cells express high levels of EGFR 12, we hypothesized that the ability of cetuximab to mediate ADCC activity of Fcγ-CR T cells against ECCs is associated with EGFR expression level on target cells. To test this hypothesis, we measured EGFR expression level on the surface of HCT116, A549, MDA-MB-231, and MDA-MB-468 cells and correlated it with the ability of cetuximab to mediate Fcγ-CR T cell cytotoxicity with target cells (fig. 6). As expected, MDA-MB-468 cells displayed the highest MFI upon staining with fluorochrome-labeled anti-EGFR mAb (fig. 6A). Furthermore, the ability of both CD16158F-CR and CD32131R-CR to reduce the viability of EGFR+ ECCs, in combination with cetuximab, displayed a highly significant correlation with the MFI of the target marker by the cancer cell lines tested (fig. 6B).
DISCUSSION
Rituximab and trastuzumab have been utilized to redirect first and second generation CD16158V-CR T cells against CD20+ and HER2+ hematologic and solid malignancies, respectively 7,9,10. The results obtained in pre-clinical studies suggest that CD16158V-CR T cells may act as universal chimeric receptor-effector cells capable of improving therapeutic effectiveness of TA-specific mAbs by an ADCC mechanism. The rationale, underlying the choice of the high-affinity extracellular CD16158V, for manufacturing Fc chimeras, is that CD16 triggers ADCC in NK cells 13. However, myeloid cells such as monocyte/macrophages and granulocytes can also mediate effector functions such as proinflammatory cytokine production 14,15 and cell-mediated cytotoxicity 16 including ADCC 17,18. Although CD16 is the major player in mediating ADCC, CD32 is also capable of promoting ADCC by myeloid cells 17. Similarly to CD16, CD32 is characterized by low (CD32131R) and high affinity (CD32131H) polymorphisms 2.
To date, there is scant information about the anti-tumor activity of CD16158F-CR T cells and the role of CD32131R-CR T cells is completely unknown. Growing experimental evidence suggests that CD16158F and CD32131R polymorphisms show differential binding affinities for IgG1 and IgG2 mAb Fc portions. Taking advantage from the availability of cetuximab (IgG1) and panitumumab (IgG2), here we demonstrate, for the first time, that both CD32131R- and CD16158F-CR T cells trigger ADCC to the TNBC cells, MDA-MB-468. In a side by side comparison, we show the superiority of CD32131R-CR over CD16158F-CR in redirecting engineered T cells against the MDA-MB-468 cells through anti-EGFR mAbs at least in vitro.
The transduction frequency of a retroviral vector encoding CD32131R-CR was significantly higher than that of CD16158F-CR. The higher expression frequency of CD32131R-CR on T cells may reflect the preferential propensity of hematopoietic cells to express CD32 as compared to CD16 19. This result may be of relevance for manufacturing high numbers of selected engineered T cells for in vivo pre-clinical and clinical studies.
The affinity of CD16158F-CR and CD32131R-CR for IgG1 and IgG2 mAbs was significantly different. CD32131R-CR bound Fc fragments of soluble IgG1 (anti-CD107A and cetuximab) and to a lesser extent IgG2 (panitumumab) while CD16158F-CR T cells bound neither. The differential binding ability of cetuximab and panitumumab to CD32131R-CR is not surprising since CD32131R polymorphisms bind IgG1 with a significantly higher affinity than IgG2. On the other hand, the failure of CD16158F-CR to bind soluble cetuximab (IgG1) is surprising since CD16158F polymorphism has slightly lower ability to bind IgG1 than CD32131R 2. Our results are a bit different from those of Kudo et al. who showed a weak but detectable binding of soluble IgG1 mAbs, such as rituximab and trastuzumab, to CD16158F-CR. The different results obtained by Kudo et al 7 and by ourselves may reflect structural differences between the CD16158F-CR endodomain.
The induction of an effective ADCC of tumor cells by FcγR+ cytotoxic T cells needs to meet at least three distinct criteria. They include i) the presence of FcγR+ cytotoxic T cells with a functional lytic machinery; ii) FcγR binding affinity for the tested mAb Fc fragment sufficient to activate T cells; and iii) surface expression level of the antigen targeted by the tested mAb sufficient to activate effector mechanisms in T cells. Indeed, both CD16158F-CR and CD32131R-CR engineered T cells fully satisfy the first condition since anti-CD16 and anti-CD32 mAbs triggered a similar level of reverse ADCC when tested with KRAS-mutated, FcγR positive, HCT116 cells. However, CD16158F-CR T cells and CD32131R-CR T cells, in combination with cetuximab, neither released IFNγ and TNFγ and TNFα nor eliminated KRAS mutated ECCs including A549, HCT116, and MDA-MB-231 cells 20,21, although they were fully activated by the EGFR overexpressing MDA-MB-468 cells.
The differential antitumor activity of both Fcγ-CR T cells with wild type and KRAS-mutated ECC cells deserves some comments. The inability of both Fcγ-CR T cells to eliminate KRAS-mutated ECC cells does not reflect a mechanism of resistance of these target cells to the lytic activity of the two effector cells tested, since both of them can eliminate KRAS-mutated HCT116FcγR+ cancer cells in a redirect ADCC assay. On the other hand, the higher sensitivity of the wild type MDA-MB-468 than of the tested KRAS-mutated ECCs is likely to reflect the differential levels of surface EGFR expression. Indeed, the KRAS wild type, MDA-MB-468 cells overexpress EGFR 22 at a level higher than that on the KRAS-mutated ECCs utilized in this study. Our hypothesis is supported by Derer et al.’s finding that a KRAS mutation impairs the sensitivity of CRC cells to anti-EGFR mAbs because of C/EBPβ-dependent downregulation of EGFR expression 23.
The restoration of the sensitivity of KRAS-mutated ECCs to Fcγ-CR T cell lytic activity may require the generation of CD32-CR and CD16-CR with high affinity for the used mAb Fc fragments such as CD32131H-CR and CD16158V-CR. This strategy is supported by the ability of ECCs opsonized with anti-EGFR mAbs to induce a level of FCγR cross-linking insufficient to fully mediate ADCC, but sufficient to stimulate other FCγ-CR T cell functions such as cytokine production. In its support, we show that HCT116 and A549 cells, opsonized with panitumumab, promote IFNγ and TNFαrelease from CD32131R-CR T cells.
As a consequence, failure to generate an effective ADCC could be related to inadequate expression of EGFR on the surface of ECCs. In support of this hypothesis, CD16158F-CR T and CD32131R-CR T cells in combination with cetuximab damaged wild-type MDA-MB-468 cells overexpressing EGFR. Indeed, the results shown in figure 6 indicate that the extent of ECC reduced viability induced by the combination of either CD16158F-CR T cells or CD32131R-CR T cells with cetuximab directly correlated with EGFR expression levels on target ECCs. EGFR cross-linking MDA-MB-468 cells, with Fcγ-CR T cells and cetuximab, led to ADCC activation. The ability of CD16158F-CR T cells to mediate ADCC in the presence of cetuximab is somewhat unexpected since no binding of soluble cetuximab to these cells could be detected. This finding may reflect the ability of CD16158F-CR T cells to bind cetuximab only after EGFR cancer cell opsonization, which stabilizes ligand-receptor interactions.
Unlike cetuximab (IgG1), panitumumab (IgG2) did not induce significant ADCC by NK cells limiting its applications in cell-based cancer immunotherapy 24. However, panitumumab is still able to trigger ADCC by macrophages, which, with the exception of a small subset of cells 25, do not express CD16 but express CD32 and CD64 26. As a logical consequence, engineering cytotoxic T cells with a CD32131R-CR has allowed us to demonstrate that panitumumab can stimulate strong ADCC by CD32131R-CR T cells against MDA-MB-468 cells overexpressing EGFR. These results open new perspectives for the use of panitumumab in cell-based targeted immunotherapy of solid tumors.
ACKNOWLEDGMENTS
This work was supported by the Italian Association for Cancer Research (AIRC) under grant IG17120. We thank Spagnoli G.C., Coccia M., and Rossi A. for technical support and Dr. Paggiolu M. and Dr. Papa P. for administrative assistance.
Footnotes
Disclosure of Potential Conflicts of Interest: The authors declare no potential conflict of interest.
Abbreviations
- ADCC
- antibody-dependent-cellular-cytotoxicity
- APC
- allophycocyanin
- BC
- breast cancer
- CM
- complete medium
- CR
- chimeric receptor
- CRC
- colorectal carcinoma
- DMEM
- Dulbecco’s Modified Eagle’s Medium
- ECCs
- EGFR positive epithelial cancer cells
- EGFR
- epidermal growth factor receptor
- FBS
- fetal bovine serum
- FcR BR
- Fc receptor blocking reagent
- FITC
- fluorescein isothiocyanate
- IMDM
- Iscove’s Modified Dulbecco’s Medium
- IL-7
- interleukin-7
- IL-15
- interleukin-15
- INFγ
- interferon gamma
- mAb
- monoclonal antibody
- MFI
- mean fluorescence intensity
- NSCLC
- non-small cell lung cancer
- PBMCs
- peripheral blood mononuclear cells
- Pe
- phycoerythrin
- RT-PCR
- reverse-transcriptase polymerase chain reaction
- DMSO
- dimethyl sulfoxide
- OD
- optical density
- TA
- tumor antigen
- TNBC
- triple negative breast cancer
- TNFα
- tumor necrosis factor alpha