CD16-158-Valine Chimeric Receptor T Cells Overcome the Resistance of KRAS-mutated Colorectal Carcinoma Cells to Cetuximab

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][7][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][10][11][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.

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 CD32 131R -, CD32 131H -, CD16 158F -or CD16 158V -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 (0.5x10 6 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% CO 2 atmosphere. After transduction, T cells were expanded in RPMI 1640 CM supplemented with 10 ng/ml IL-7 and 5 ng/ml IL-

In vitro tumor cell viability assays
Antitumor activity of Fcγ-CR T cells in vitro was evaluated by MTT assays. Tumor target cells (7x10 3 /well) were seeded in 96-well plates and Fcγ-CR T cells (35x10 3 /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 (1x10 5

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. Diseasefree 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 CD32 131H -CR (83.5%±9.5%) and CD32 131R -CR (77.7%±13.2%) was significantly more effective than that of CD16 158V -CR (51.7%±13.7%) and CD16 158F -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 1 0 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. 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 (CD16 158V/V ) and presence of histidine instead of arginine at position 131 of CD32 (CD32 131H/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. CD16 158V has preferentially been utilized for in vitro and in vivo studies [9][10][11]23 . However, Kudo et al.
showed that CD16 158V -CR has significantly higher affinity for rituximab than CD16 158F -CR.
Here, neither CD16 158V -CR nor CD16 158F -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. CD32 131R -CR bound soluble cetuximab and panitumumab with higher affinity than CD32 131H -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. 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 CD16 158V -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, 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 CD16 158V -CR T cells was then assessed. Transduced cell ability to impair KRAS-mutated HCT116 viability was tested prior to their administration to experimental animals. CD16 158V -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 CD16 158V -CR T cells, combined with cetuximab, significantly affected HCT116 cell viability. Instead, CD16 158V -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 CD16 158V -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. CD16 158V -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 CD16 158V -CR. These results indicate that CD16 158V -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 CD16 158V -CR combined with cetuximab can control KRAS-mutated cancer cell growth in vitro and in vivo. Our results by 1 3 using the same KRAS-mutated HCT116 cells and at an equivalent reverse ADCC potential of Fcγ-CR utilized, the superior activity of CD16 158V -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   HCT116 cells were incubated for 72h, at 37°C, in the presence or absence of CD16 158V -CR T cells at the indicated E: T ratios with or without cetuximab (C) or panitumumab (P) both at the concentration of 3μg/ml. Non-transduced T cells were used as a control. 1 6 HCT116 cell viability was evaluated by MTT assay. Values are expressed as mean± SD.
Data are analyzed by a two-way ANOVA test. Panel B: four groups of CB17 SCID mice (N=5 per group) were injected with HCT116 cells (1×10 6 ) subcutaneously, in the right flank. Following HCT116 injection, three groups of mice were injected, in the area adjacent to HCT116 injection, with 150 μ g of cetuximab (group 2), 5x10 6 CD16 158V -CR T cells