A potential therapy for chordoma via antibody-dependent cell-mediated cytotoxicity (ADCC) employing NK or high affinity NK (haNK) cells in combination with cetuximab

Abstract

OBJECTIVE:

Chordoma is a rare bone tumor derived from the notochord, and is resistant to conventional therapies such as chemotherapy, radiotherapy, and targeting therapeutics. Expression of epidermal growth factor receptor (EGFR) in a large proportion of chordoma specimens indicates that chordoma may be a potential target for therapeutic intervention. This study investigated the potential role of the anti-EGFR antibody cetuximab in the immunotherapy for chordoma.

METHODS:

Since cetuximab is a monoclonal antibody of the IgG1 isotype, it has the potential to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) employing natural killer (NK) cells as effectors. Polymorphisms in the CD16 allele expressed on NK cells has been shown to influence the degree of ADCC of tumor cells, with the high affinity valine (V)/V allele being responsible for more lysis than the V/phenylalanine (F) or FF allele. Unfortunately, however, only approximately 10% of the population expresses the VV allele on NK cells. An NK cell line, NK-92, has now been engineered to endogenously express IL-2 and the high affinity (ha) CD16 allele. These irradiated high-affinity cells were analyzed for lysis of chordoma cells with and without cetuximab, and the levels of lysis observed in ADCC were compared with those of NK cells from donors expressing the VV, VF, and FF alleles.

RESULTS:

Here we demonstrate for the first time (a) that cetuximab in combination with NK cells can mediate ADCC of chordoma cells; (b) the influence of the NK CD16 polymorphism in cetuximab-mediated ADCC for chordoma cell lysis; (c) that engineered high-affinity (ha) NK (haNK) cells, i.e., cells transduced to express the CD16 V158 FcγRIIIa receptor, bind cetuximab with similar affinity to normal NK cells expressing the high affinity VV allele; and (d) that irradiated haNK cells induce ADCC with cetuximab in chordoma cells.

CONCLUSIONS:

These studies provide the rationale for the use of cetuximab in combination with irradiated haNK cells for the therapy of chordoma.

INTRODUCTION

Chordoma, a rare bone tumor, is thought to be derived from residual notochord. Accounting for 20% of primary spinal tumors and only 1%–4% of all malignant bone tumors, approximately 300 new cases per year are diagnosed in the United States ^7,43, with approximately 2400 patients alive with chordoma in the U.S. annually. The median overall survival from time of diagnosis is an estimated 6–7 years. ²⁵ Surgery followed by radiation therapy is the usual “standard of care,” but the anatomic location and size of the tumor often prevent curative excision with clear margins. Thus, relapse is common and metastases have been reported in up to 40% of cases. No agent has been approved by the U.S. Food and Drug Administration for chordoma therapy since it is largely resistant to standard cytotoxic chemotherapy ³⁷, creating an urgent need for novel therapeutic modalities for chordoma.

Cytotoxic chemotherapy’s ineffectiveness in chordoma has spurred increasing efforts to identify new therapeutic modalities. mTOR, b-type platelet-derived growth factor receptor, vascular endothelial growth factor, and epidermal growth factor receptor (EGFR) have been identified as potentially relevant therapeutic targets for chordoma. ¹⁸ Previous studies have shown that a large proportion of chordomas express EGFR. ^35,38,44 Cetuximab, an IgG1 anti-EGFR monoclonal antibody (MAb), is the only anti-EGFR agent that both blocks the EGFR-dependent proliferation pathway and has the potential to induce antibody-dependent cell-mediated cytotoxicity (ADCC). ¹² In in vitro studies, cetuximab mediated ADCC in several types of cancer cells that express EGFR, including esophageal cancer, non-small cell lung cancer, and squamous cell carcinoma of the head and neck. ²⁷ Several therapeutic agents targeting EGFR, including erlotinib, gefitinib, lapatinib, and sapatinib, have been shown to inhibit proliferation of chordoma cells. ^34,36 To date, however, employing radiation and/or these and other agents, the response rate for patients has been extremely low, i.e., less than 5%. The potential of cetuximab-mediated ADCC in chordoma has not previously been investigated.

ADCC is mediated by the binding of a human IgG1 antibody with its ligand on tumor cells, and with the CD16 Fc receptor on NK cells. Interaction between IgG1 antibody-bound tumor cells and Fcγ receptor triggers the activation and degranulation of the NK cells (Figure 1). NK cells from healthy donors can express three type of polymorphism in the CD16 allele; a) endogenous alleles CD16 valine (V) high affinity Fc receptor FcγRIIIa(158V) only (V/V genotype), b) the lower affinity phenylalanine (F) allele only (F/F genotype), or c) express both (V/F genotype). In general, the NK cells of the VV allele are the most efficient effectors in ADCC. Unfortunately, only approximately 14% of humans express the VV allele on NK cells (Figure 1). ^{8,26,30,31,41,45,46} An NK cell line derived from a lymphoma patient has been shown, as an irradiated adoptively transferred agent, to be safe and has provided preliminary evidence of clinical benefit. ^2,15,40 The NK-92 cell line, however, does not express CD16 and also requires IL-2 for propagation. The NK-92 cell line, devoid of CD16, has now been engineered to express the high affinity (ha) CD16 V158 FcγRIIIa receptor, as well as engineered to express IL-2, and is designated haNK. ¹⁴

Figure 1: Model of proposed mechanism of natural killer (NK) cell mediated antibody-dependent cellular cytotocicity (ADCC).

A. Chordoma cells express EGFR. The anti-EGFR monoclonal antibody cetuximab (humanIgG1) binds EGFR. B. The Fc portion of the cetuximab is bound by the CD16 receptor of NK cells, forming a bridge that triggers granzyme degranulation and chordoma cell lysis (A). C. Patient NK cells express polymorphic CD16 receptors that bind antibody Fc at different affinities. The strongest CD16 affinity, VV is seen in 14% of the population, while the lower affinity CD16 receptors VF and FF are seen in 82% of the population. To compensate for potentially lower affinity CD16 receptor bearing endogenous NK cells, high affinity NK cells (haNK; NK cells engineered to express high affinity CD16 receptor and IL-2) can be infusion into patients.

Here we demonstrate for the first time (a) that cetuximab in combination with NK cells can mediate ADCC of chordoma cells; (b) the influence of the NK CD16 polymorphism in cetuximab-mediated ADCC for chordoma cell lysis; (c) that engineered high-affinity (ha) NK (haNK) cells, i.e., cells transduced to express the CD16 V158 FcγRIIIa receptor, bind cetuximab with similar affinity to normal NK cells expressing the high affinity VV allele; and (d) that irradiated haNK cells induce ADCC with cetuximab in chordoma cells. Our findings suggest that while chordoma responds poorly to conventional therapies, the combination of adoptively transferred irradiated haNK cells plus cetuximab may have clinical benefit for chordoma patients (Figure 1).

METHODS

Cell culture and reagents

The chordoma cell lines JHC7 and UM-Chor1 were obtained from the Chordoma Foundation (Durham, NC). The chordoma cell lines U-CH2 (ATCC® CRL-3218 ™) and MUG-Chor1 (ATCC® CRL-3219 ™) were obtained from American Type Culture Collection (Manassas, VA). All cell lines were passaged for fewer than 6 months and were maintained as previously described ¹¹. haNK cells were provided through a Cooperative Research and Development Agreement (CRADA) between the NCI and NantBioScience (Culver City, CA). haNK cells were cultured in phenol-red free and gentamycin-free X-Vivo-10 medium (Lonza, Walkersville, MD) supplemented with 5% heat-inactivated human AB serum (Omega Scientific, Tarzana, CA) at a concentration of 5×10⁵/ml. haNK cells were irradiated with 10 Gy 24 h before all experiments. Peripheral blood mononuclear cells (PBMCs) from healthy volunteer donors were obtained from the NIH Clinical Center Blood Bank (NCT00001846).

Flow cytometry

Antihuman MAbs used were as follows: PE-EGFR (BD Biosciences, San Jose, CA), FITC-CD16 clone 3G8 (BD Biosciences), APC-CD56 (BioLegend, San Diego, CA), PE-CD226 (DNAM-1) (BD Biosciences), PerCP-Cy5.5-NKG2D (BD Biosciences), PE-Cy7-perforin (eBioscience, San Diego, CA). Samples were acquired on a FACSCalibur flow cytometer or FACSVerse (Becton Dickinson, Franklin Lakes, NJ) and analyzed using FlowJo software (TreeStar, Inc., Ashland, OR). Isotype control staining was < 5% for all samples analyzed.

Antibody-dependent cellular cytotoxicity assay

The ADCC assay was performed as previously described ⁵, with indicated modifications. NK effector cells were isolated from normal donor PBMCs using the Human NK Cell Isolation (negative selection) Kit 130-092-657 (Miltenyi Biotec, San Diego, CA) following the manufacturer’s protocol, resulting in > 80% purity, and allowed to rest overnight in RPMI-1640 medium containing 10% fetal bovine serum. Tumor cells were harvested and labeled with ¹¹¹In. Cells were plated as targets at 2,000 cells/well in 96-well round-bottom culture plates and incubated with 10 μg/mL of cetuximab (Erbitux®; Lilly, Indianapolis, IN) or unresponsive rituximab (Rituxan®; Biogen, Cambridge, MA) as a control isotype antibody at room temperature for 30 min. NK cells or haNK cells were added as effector cells. Various effector:target cell ratios were used in the study. After 4 h or 20 h, supernatants were harvested and analyzed for the presence of ¹¹¹In using a WIZARD2 Automatic Gamma Counter (PerkinElmer, Waltham, MA). Spontaneous release was determined by incubating target cells without effector cells, and complete lysis was determined by incubation with 0.05% Triton X-100 (Sigma-Aldrich, St. Louis, MO). Experiments were carried out in triplicate. Specific ADCC lysis was determined using the following equation: Percent lysis = [(experimental cpm − spontaneous cpm) / (complete cpm − spontaneous cpm)] × 100.

To verify that CD16 (FcγRIII) on NK cells engages ADCC lysis mediated by cetuximab, CD16 MAb was used to block CD16. NK cells were incubated with 2 μg/mL of CD16 MAb (clone B73.1; eBioscience) and haNK cells were incubated with 50 μg/mL of CD16 MAb for 2 h before being added to target cells.

CD16 (FcγRIIIa) genotyping

DNA was extracted from PBMCs of healthy donors using a QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA), and stored at −80°C until use. The polymorphism of CD16 at amino acid position 158 that is a valine (V) vs. phenylalanine (F) was determined using allele-specific droplet digital polymerase chain reaction (PCR) employing the TaqMan array for CD16 (rs396991; Life Technologies, Waltham, MA). ^24,26,32 A master reaction mix was prepared, and 1 μL of genotyping DNA was added. The PCR reaction was performed on a Bio-Rad T100 thermal cycler (Bio-Rad, Hercules, CA) for 40 cycles at 95°C for 10 min, 94°C for 30 sec, and 60°C for 1 min. The plate was read on a Bio-Rad QX200 droplet reader. Data were analyzed with Bio-Rad QuantaSoft v.1.5 software.

Cetuximab-binding properties of FcγRIIIa

Cetuximab-binding properties of FcγRIIIa were examined as previously reported ⁸, with indicated modifications. NK cells or haNK cells were incubated with varying concentrations of cetuximab at 4°C for 30 min. Cells were then incubated with FITC-conjugated CD16 3G8 at 4°C for 30 min, washed twice with 1% BSA/PBS, and analyzed by flow cytometry. Results were expressed as the percentage of inhibition of CD16 3G8 binding: (MFI in absence of cetuximab − MFI in the presence of cetuximab) × 100/(MFI in the absence of cetuximab).

Statistical analysis

Significant differences in the distribution of data acquired by ADCC assays were determined by paired Student’s t test with a 2-tailed distribution and reported as P values, using Prism 6.0f software (GraphPad Software Inc., La Jolla, CA). A P value < 0.05 was significant.

RESULTS

Cetuximab increases NK-cell lysis of chordoma cell lines via ADCC

It has previously been shown that chordoma cell lines express EGFR ³⁴. We qualitatively confirmed and extended this finding employing four human chordoma cell lines: JHC7, UM-Chor1, U-CH2, and MUG-Chor1 (Figure 2). The 4 chordoma cell lines express between 13% to 80% EGFR as determined by flow cytometry, although the absolute expression levels of EGFR can modulate with tissue culture density and time in culture.

Figure 2: EGFR expression in chordoma cell lines.

Inset numbers indicate % positive cells and mean fluorescence intensity (MFI) (parentheses).

We next performed an in vitro assay to determine cetuximab-mediated ADCC in chordoma cell lines employing NK cells from healthy donors as effectors. As shown in Figure 3A, cetuximab significantly increased NK-cell lysis relative to the isotype control antibody in JHC7 cells (13.7-fold; P < 0.01), UM-Chor1 cells (10.5-fold; P < 0.01), U-CH2 cells (83.5-fold; P < 0.01), and MUG-Chor1 cells (59-fold; P < 0.01). Cetuximab alone (no NK cells) did not mediate lysis of chordoma cells (data not shown). NK-cell lysis via ADCC occurs when CD16 (FcγRIII) on NK effector cells interacts with the Fc portion of antibodies recognizing target cells (Figure 1). ¹ As shown in Figure 3B, the addition of CD16 neutralizing antibody inhibited cetuximab-enhanced NK-cell lysis in both the JHC7 and UM-Chor1 cell lines analyzed, indicating that cetuximab-induced NK-cell lysis was mediated by ADCC.

Figure 3: Cetuximab increased NK-cell lysis via ADCC in chordoma cell lines.

A. ADCC assays were performed using four chordoma cell lines, using normal donor NK cells at an effector:target (E:T) ratio of 20:1. Indicated groups were incubated with cetuximab. B. ADCC assays were performed using two chordoma cell lines, using normal donor NK cells at an E:T ratio of 20:1. Indicated groups were incubated with cetuximab and anti-CD16 antibody. Statistical analyses were done by Student’s t-test, * = P < 0.01, error bars indicate mean ± S.D. for triplicate measurements. This experiment was repeated at least two times with similar results.

CD16 polymorphism of NK cells is associated with cetuximab-mediated ADCC of chordoma cells

The CD16 (FcγRIIIa) polymorphism expressed on NK cells is associated with affinity of CD16 for IgG1 MAbs (Figure 1). ^8,17 The FCGR3A gene, which encodes FcγRIIIa, displays a functional allelic dimorphism that generates allotypes with either an F or a V residue at amino acid position 158. ³² Previous studies have shown that the magnitude of response to cetuximab-mediated ADCC is related to FcγRIIIa polymorphism of NK cells in head and neck squamous cell carcinoma cells. ^22,39 We next performed in vitro assays for ADCC activity mediated by cetuximab using FCGR3A-genotyped normal donor NK cells that expressed the FcγRIIIa-158 FF, VF, or VV allele. With control isotype antibody, UM-Chor1 cells were killed at very low levels by NK cells regardless of NK phenotype (Figure 4A). However, cetuximab increased NK-cell lysis in all the NK-cell phenotypes. Cetuximab-induced lysis by NK cells from three donors expressing the FcγRIIIa-158 FF allele was 24%, 17%, and 15%, respectively. In contrast, cetuximab-induced ADCC lysis by NK cells using three VF donors was 34%, 49%, and 32%, respectively, and 51%, 66%, and 59% lysis, respectively, using NK cells from three VV donors. There was a significant positive correlation (R² = 0.85) for the mean of cetuximab-mediated ADCC lysis induced by NK cells from three FF (19%), three VF (38%), three VV (59%) donors (Figure 4B). Taken together, these results suggest that NK cells that express the FcγRIIIa-158 V allotype exhibit enhanced cetuximab-mediated ADCC in chordoma cells.

Figure 4: Cetuximab-mediated ADCC by CD16 polymorphism-genotyped NK cells.

A. ADCC assays were performed using UM-Chor1, using NK cells from three FF, three VF, and three VV normal donors at an E:T ratio of 10:1. Indicated groups were incubated with cetuximab. B. Correlation of cetuximab-mediated ADCC with NK cells from three FF, three VF, and three VV donors. Statistical analyses were done by Student’s t-test, * = P < 0.05, error bars indicate mean ± S.D. for triplicate measurements.

The phenotype of CD16 polymorphism-genotyped NK cells and haNK cells

NK cells from some individuals can be potent cytotoxic effectors for cancer therapy. However, there can be technical challenges to obtaining sufficient numbers of functionally active NK cells from patients. As an alternative, several cytotoxic NK cell lines have been generated, including NK-92 These NK-92 cells, designated hank (Figure 1) have recently been engineered to endogenously express IL-2 and the high affinity (ha) CD16 V158 FcγRIIIa receptor (designated haNK). ^14,16 We compared the phenotype (CD56, DNAM-1, NKG2D, perforin, and CD16) of CD16 polymorphism-genotyped normal donor NK cells with that of haNK cells. While there were only minor differences in the percentage of cells expressing a given marker, there were substantial differences observed in the levels of expression as determined by mean fluorescence intensity (MFI). Compared to NK VV donors, haNK cells had a 20-fold higher MFI of CD56 (Figure 5A), 2.9-fold higher expression of DNAM-1 (Figure 5B), and 1.8-fold higher expression of NKG2D (Figure 5C). There was no difference in perforin expression between NK cells and haNK cells (Figure 5D). The mean MFI of CD16 was 1.5-fold higher in VV donors compared to FF donors and haNK cells (Figure 5E).

Figure 5: The phenotype of CD16 polymorphism-genotyped NK cells and haNK cells.

Expression levels of A. CD56, B. DNAM-1, C. NKG2D, D. perforin, and E. CD16 of NK cells from three FF, three VF, three VV normal donors and haNK cells. Numbers indicate % positive cells (upper panels) and MFI (lower panels). Bars indicate the mean.

haNK cell–mediated ADCC of chordoma cells with cetuximab

To examine the potential utility of haNK cells for cetuximab therapy of chordoma, we performed an in vitro assay for cetuximab-mediated ADCC using haNK cells as effectors (Figure 6A). Lysis by haNK cells with isotype control was 11.8% of JHC7 cells and 2.6% of UM-Chor1 cells. Cetuximab significantly enhanced haNK-cell lysis compared to isotype control in both JHC7 (1.7-fold; P < 0.01) and UM-Chor1 cells (2.6-fold; P < 0.01). The addition of CD16 neutralizing antibody inhibited cetuximab-enhanced haNK-cell lysis in both JHC7 and UM-Chor1 cell lines (data not shown). As NK cells have previously been shown to be “serial killers,” i.e., one NK cell can lyse up to five target cells ^3,42, 20-h ¹¹¹In-release assays were also carried out (Figure 6B). There, the lysis of the two chordoma cell lines was markedly greater. These results indicate that haNK cells induce ADCC via cetuximab in chordoma cells. To determine relative affinities, we next compared the ability of cetuximab to inhibit the binding of FITC-conjugated CD16 MAb to CD16 polymorphism-genotyped normal donor NK cells and haNK cells (Figure 7A). A 50% inhibition of CD16 Ab binding to NK cells from four FF donors was achieved with 220 μg/mL of cetuximab. Compared to FF donors, a 4.5-fold lower (49.2 μg/mL) and 2.8-fold lower (80 μg/mL) concentration of cetuximab showed a 50% inhibition of CD16 Ab binding to normal NK cells from VV donor and haNK cells, respectively (Figure 7B). These results show that both NK cells expressing FcγRIIIa-158 VV and haNK cells bind cetuximab with higher affinity than NK cells expressing FcγRIIIa-158 FF.

Figure 6: Cetuximab increased haNK-cell lysis via ADCC in chordoma cell lines.

ADCC assays were performed using two chordoma cell lines, using haNK cells as effector cells at an E:T ratio of 20:1 for A. 4h and B. 20h. Indicated groups were incubated with cetuximab and/or anti-CD16 antibody. Statistical analyses were done by Student’s t-test, * = P < 0.01, error bars indicate mean ± S.D. for triplicate measurements. This experiment was repeated at least two times with similar results.

Figure 7: CD16 affinity to cetuximab of NK cells vs. haNK cells.

NK cells from four FF and two VV normal donors and haNK cells were incubated with varying concentrations of cetuximab, followed by FITC-conjugated CD16 Ab. Percentages of inhibition of CD16 MAb binding were calculated as described in Materials and Methods. A. Percentages of inhibition of CD16 MAb binding shown by each donor. B. The mean of percentages of inhibition of CD16 MAb binding.

DISCUSSION

Based on preclinical evidence of the role of EGFR in chordoma pathogenesis and the observation by immunohistochemistry that over 70% of chordoma specimens express EGFR ³⁵, several clinical trials targeting EGFR have been undertaken in chordoma. ¹⁸ However, because these trials were not randomized or well controlled, no consensus has been reached concerning the therapeutic benefit of EGFR inhibition in chordoma. In two separate case reports, the combination of the EGFR MAb cetuximab and gefitinib, a tyrosine kinase inhibitor of EGFR, achieved partial radiographically defined responses. ^13,20 However, the potential of cetuximab-induced ADCC in chordoma was not directly examined. Here, we show that cetuximab markedly and significantly increased NK-cell lysis via ADCC in four of four chordoma cell lines (Figure 3). Moreover, NK cells obtained from healthy donors carrying FCGR3A-158 VV induced higher cetuximab-mediated ADCC lysis in chordoma (Figure 4). Previous in vitro studies have indicated that the FcγRIIIa-158 VV phenotype of NK cells enhances the affinity of CD16 to IgG1 ¹⁴, inducing ADCC mediated via IgG1 MAb in several types of cancer cell lines. ^8,14,29 Some clinical studies have also shown that FcγRIIIa polymorphisms of NK cells correlated with response to IgG1 MAb therapy. Musolino et al. reported that patients with metastatic breast cancer who had FCGR2A-131 HH and/or FCGR3A-158 VV genotypes had a significantly better objective response rate and progression-free survival with trastuzumab therapy than patients with neither genotype. ²⁹ In a study of 49 patients with follicular lymphoma, FCGR3A-158 VV patients had an improved response to rituximab. ⁶ Three retrospective studies in metastatic colorectal cancer patients treated with cetuximab reported that VV is the most beneficial FCGR3A-158 genotype. ^4,10,33 Taken together, our observations indicate that cetuximab has potential clinical benefit for chordoma patients, especially in those 14% of patients with the FCGR3A-158 VV genotype or in combination with haNK cell infusion.

Although ADCC induction can be observed in in vitro models, clinical translation raises some obstacles. First, recruiting sufficient numbers of functionally active NK cells to tumor tissues is technically challenging since they represent only 10% of lymphocytes, and are often dysfunctional in a cancer-induced immunosuppressive environment. ⁹ Moreover, chemotherapy and radiation therapy, first-line treatment for metastatic/advanced chordoma, could also reduce the number and activity of lymphocytes. Adoptive NK-cell therapies have been developed to supply sufficient numbers of functional NK cells for patients. The cytotoxic NK-92 cell line was generated for adoptive transfer therapy from a 50-year-old male patient with progressive non-Hodgkin’s lymphoma. Four phase I trials in different malignancies have been conducted using irradiated NK-92 cells. The infusions were well tolerated, and clinical responses were observed in patients with hematological malignancies, melanoma, lung cancer, and kidney cancers. Since NK-92 cells do not express the FcγRIIIa receptor, they cannot mediate ADCC. The NK cell line designated haNK was established by inducing high-affinity CD16, V158 FcγRIIIa receptor to NK-92 cells. ¹⁵ Since only approximately 14% of the population is homozygous for the high-affinity FcγRIIIa receptor (FCGR3A-158 VV) ^19,21, there is a clear rationale for infusing haNK cells into patients who carry the genotype of low- or intermediate-affinity FcγRIIIa receptor to maximize MAb efficacy. Our results show that haNK cells have 2.8-fold higher affinity to cetuximab than NK cells from healthy donors carrying FCGR3A-158 FF (Figure 7B). Consistent with their high binding ability to cetuximab, haNK cells significantly induced ADCC via cetuximab in chordoma cells (Figure 6). Moreover, since 10⁹ to 10¹⁰ irradiated NK-92 cells were shown to be safely administered to cancer patients, the potential exists for similar levels of adoptive transfer of irradiated haNK cells, even in patients whose endogenous NK cells express the VV phenotype.

NK-92 cells have been shown to express large numbers of activating receptors such as NKp30, NKp46, and NKG2D. ²³ NKG2D and DNAM-1 are the best-characterized activating NK-cell receptors implicated in immune response against cancers. Both receptors recognize their ligands expressed on tumor cells and induce target-cell lysis. ²⁸ Our data show that haNK cells have higher expression of NKG2D and DNAM-1 by MFI compared to normal NK cells (Figure 5A and B), indicating a greater ability to recognize and lyse tumor cells. Without cetuximab, NK cells from healthy donors lysed chordoma cells at extremely low levels (Figures 3 and 4). In contrast, haNK cells induced greater lysis of chordoma cells without cetuximab (Figure 6B).

Here we show for the first time that cetuximab can induce ADCC in chordoma cells, while chordoma cells were not killed in significant numbers by NK cells alone. Moreover, NK cells that express FcγRIIIa-158 VV induced higher cetuximab-mediated ADCC of chordoma cells. An engineered NK-92 cell line, transduced with high-affinity FcγRIIIa (haNK cells), bound cetuximab with high affinity, resulting in haNK cell-induced ADCC via cetuximab of chordoma cells. Our results also indicate that cetuximab therapy could lead to a better clinical outcome for chordoma patients who have NK cells expressing the CD16 V158 FcγRIIIa receptor allele. Adaptively transferred irradiated haNK cells could provide sufficient numbers of functional NK cells for all chordoma patients and could functionally convert FCGR3A-158 FF carriers to VV carriers. Our findings provide the rationale that cetuximab plus irradiated haNK cell-mediated immunotherapy may have potential clinical benefit for patients with chordoma.