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Keywords:

  • ADCC;
  • cetuximab;
  • Fcγ receptor;
  • GM allotypes;
  • trastuzumab

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Antibody-dependent cell-mediated cytotoxicity (ADCC), which links the innate and the adaptive arms of immunity, is a major host immunosurveillance mechanism against tumours, as well as the leading mechanism underlying the clinical efficacy of therapeutic antibodies such as cetuximab and trastuzumab, which target tumour antigens, human epidermal growth factor receptor (HER)1 and HER2, respectively. Immunoglobulin (Ig)G antibody-mediated ADCC is triggered upon ligation of Fcγ receptor (FcγR) to the Fc region of IgG molecules. It follows that genetic variation in FcγR and Fc could contribute to the differences in the magnitude of ADCC. Genetic variation in FcγR is known to contribute to the differences in the magnitude of ADCC, but the contribution of natural genetic variation in Fc, GM allotypes, in this interaction has hitherto not been investigated. Using an ADCC inhibition assay, we show that IgG1 expressing the GM 3+, 1−, 2− allotypes was equally effective in inhibiting cetuximab- and trastuzumab-mediated ADCC of respective target cells, in the presence of natural killer (NK) cells expressing either valine or phenylalanine allele of FcγRIIIa. In contrast, IgG1 expressing the allelic GM 17+, 1+, 2+ allotypes was significantly more effective in inhibiting the ADCC – mediated by both monoclonal antibodies – when NK cells expressed the valine, rather than the phenylalanine, allele of FcγRIIIa. These findings have important implications for engineering antibodies (with human γ1 constant region) against malignancies characterized by the over-expression of tumour antigens HER1 and HER2 – especially for patients who, because of their FcγRIIIa genotype, are unlikely to benefit from the currently available therapeutics.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Antibody-dependent cell-mediated cytotoxicity (ADCC), which links the specific humoral responses to the vigorous innate cytotoxic effector responses, is a major host defence mechanism against tumours. Immunoglobulin (Ig)G antibody-mediated ADCC is triggered upon ligation of FcγR to the Fc of IgG molecules [1]. It follows that genetic variation in FcγR and Fc could contribute to the differences in the magnitude of ADCC. The contribution of the natural genetic variation in FcγR to the magnitude of ADCC against tumours expressing particular antigens has been documented in several studies [2–5], but the possible contribution of the natural genetic variation in the Fc region of IgG proteins, GM allotypes, in this interaction has not been evaluated.

A concerted effort is currently being directed at engineering Fc variants with optimized affinity for activating and inhibiting FcγRs [6,7]. The possible harmful biological consequences of increased immunogenicity of these engineered Fc variants, however, have not been investigated fully. Evaluation of the role of naturally occurring Fc (GM) variants that may have been selected evolutionarily because of their contribution (through ADCC and other protective immunosurveillance mechanisms) to survival from malignant diseases [8] is essential for engineering the next generation of humanized monoclonal antibodies, which have reduced immunogenicity, have better clinical efficacy and benefit more patients than what is possible with the currently available therapeutics.

In this investigation, we evaluated the influence of allotypically disparate IgG1 proteins on the inhibition of ADCC, in which these proteins compete with trastuzumab bound to the target SKBR-3 cells and cetuximab bound to the target A431 cells for FcγRIIIa receptors expressed on natural killer (NK) cells.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Allotyping, affinity purification and heat aggregation of IgG1 proteins

Serum samples from healthy blood donors were allotyped for all four known IgG1 allotypes – GM 1/a, 2/x, 3/f and 17/z – by a standard haemagglutination-inhibition assay [9]. The study protocol was approved by the local Institutional Review Board for human research. Total IgG from the pooled sera of subjects, 10 expressing the GM 3+, 1−, 2− and 10 expressing the alternative GM 17+, 1+, 2+ allotypes, was concentrated by ammonium sulphate fractionation. Allotypes 3 and 17 are expressed in the Fd, whereas 1 and 2 are expressed in the Fc, region of the γ1 chains. These contrasting allelic combinations provide the maximum possible allotypic differences between the two IgG1 preparations to be used in the ADCC inhibition assays. The GM allotype notation follows the international system for human gene nomenclature [10].

IgG1 proteins were isolated from the total IgG by subclass-specific affinity chromatography, and the purity of the preparation was tested by human IgG subclass-specific enzyme-linked immunosorbent assay (ELISA). Interactions between monomeric IgG and low-affinity FcγRs are unstable and require multivalent interactions for their persistence [11]. To make the interaction between the low-affinity FcγRIIIa and IgG1 more stable, the affinity-purified IgG1 was heat aggregated, following the protocol of others who have used ADCC inhibition assays [12] or have investigated IgG-FcγR interactions [13].

FcγRIIIa genotyping

A change in the nucleotide at position 559 of the FcγRIIIa gene from T to G results in amino acid change from phenylalanine (F) to valine (V) at position 158 in the IgG binding domain of FcγRIIIa. This biallelic functional polymorphism alters the level of interaction between the receptor and its IgG ligands, resulting in the modulation of the IgG-mediated effector functions [14,15]. Genotyping of FcγRIIIa alleles (dbSNP: rs396991) was performed by real-time–polymerase chain reaction (RT–PCR), using a predesigned TaqMan® genotyping assay from Applied Biosystems Inc. (Foster City, CA, USA). RT–PCR assays were performed in triplicate, using a Bio-Rad iCycler (Bio-Rad Laboratories, Hercules, CA, USA) and following the annealing and extension conditions provided in the TaqMan® assayj protocol.

Purification of NK cells

Blood from volunteers homozygous for either V or F alleles of FcγRIIIa was collected in ethylenediamine tetraacetic acid (EDTA)-coated vacutainer tubes (BD, Franklin Lakes, NJ, USA). White blood cells were isolated by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, St Louis, MO, USA). Cells were washed and cultured overnight in RPMI-1640 containing 10% fetal bovine serum (FBS) and 10% human antibody serum. NK cells were isolated by affinity depletion of non-NK cells using a kit from Milteneyi Biotec (Auburn, CA, USA), according to the manufacturer's protocol.

Cell cytotoxicity by ADCC and ADCC inhibition

This method is analogous, in principle, to that described by Macdonald et al. [12]. Human breast cancer cell line SKBR-3, which over-expresses human epidermal growth factor receptor (HER)2, and epidermal cancer cell line A431, which over-expresses epidermal growth factor receptor (EGFR)/HER1, were obtained from ATCC (Manassas, VA, USA). Cells were cultured overnight in medium [McCoy's 5A and Dulbecco's modified Eagle's medium (DMEM) for SKBR-3 and A431, respectively] containing 10% FBS, harvested with trypsin-EDTA, and washed three times with RPMI-1640 containing 1% bovine serum albumin (BSA). Cells were coated with 10 µg/ml target binding antibody (trastuzumab for SKBR-3 and cetuximab for A431) for 30 min on ice and washed three times with RPMI-1640 containing 1% BSA. (‘Extra’ trastuzumab and cetuximab were kindly provided by the Hollings Cancer Center Pharmacy.) These coated cells (5 × 103) were further incubated with 25 µg/ml of aggregated human IgG1, expressing either GM 3+, 1−, 2− or GM 17+, 1+, 2+ allotypes, and 5 × 104 NK cells (1 : 10 ratio of target to effector cells) in a volume of 100 µl RPMI-1640 containing 1% BSA in 96-well plates in triplicate for 4 h. The plates were then centrifuged and the supernatant was assayed for lactate dehydrogenase (LDH) activity using the Cytotox-96 kit from Promega Corporation (Madison, WI, USA). Spontaneous LDH release, due possibly to killer-cell immunoglobulin-like receptor dependent cytotoxicity, from target cells incubated with NK cells, was used as blank (negative control).

  • image

where the test consists of target cells incubated with aggregated IgG1, target binding antibody and NK cells and control (positive) consists of target cells incubated with target binding antibody and NK cells.

Standard deviations were obtained via experiment replication. Each experiment was performed in triplicate and repeated three times.

Statistical analysis

Student's unpaired t-test was used to compare the percentage of ADCC inhibition associated with GM-FcγRIIIa genotypic combinations. All tests were two-tailed, and the statistical significance was defined as P < 0·05.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Aggregated IgG1 proteins used in this investigation expressed two allelic phenotypes, GM 3+, 1−, 2− and GM 17+, 1+, 2+, that differ by four amino acid residues at positions 214, 356, 358 and 431 of the γ1 chain [16]. NK effector cells mediating the ADCC of HER2-expressing SKBR-3 cells and HER1-expressing A431 cells were either homozygous for the V or F allele at position 158 of the FcγRIIIa protein.

Inhibition of the binding of SKBR-3/trastuzumab complex to NK cells by GM 17+, 1+, 2+ and GM 3+, 1−, 2− allotypes of IgG1

As shown in Table 1, at a concentration of 25 µg/ml, aggregated IgG1 expressing GM 3+, 1−, 2− allotypes blocked virtually all FcγRIIIa-VV present on the NK cells, resulting in almost 100% inhibition of trastuzumab-mediated ADCC of SKBR-3 cells (97·4 ± 0·5%). This phenotype had a similar inhibitory effect on ADCC when the NK cells were homozygous for the F allele (94·5 ± 4·2%). In contrast, the inhibitory effect of IgG1 expressing the GM 17+, 1+, 2+ allotypes was significantly higher when the NK cells were homozygous for the V allele than when they were homozygous for the F allele of FcγRIIIa (73·8 ± 12·8% versus 27·8 ± 2·1%; P = 0·02). Thus it appears that, among the GM allotype-FcγRIIIa genotypic combinations investigated, IgG1 expressing the GM 17+, 1+, 2+ allotypes and homozygosity for the FcγRIIIa F allele is the least potent in inhibiting the trastuzumab-mediated ADCC of SKBR-3 cells.

Table 1.  Inhibition of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) of SKBR-3 cell by natural killer (NK) cells expressing different FcγRIIIa genotypes in the presence of allotypically disparate immunoglobulin (Ig)G1 proteins.
IgG1 phenotypeFcγRIIIa genotypeInhibition of ADCCP-value
GM 3+, 1−, 2−VV97·4 ± 0·5%0·35
FF94·5 ± 4·2%
GM 17+, 1+, 2+VV73·8 ± 12·8%0·02
FF27·8 ± 2·1%
GM 3+, 1−, 2−VV97·4 ± 0·5%0·08
GM 17+, 1+, 2+VV73·8 ± 12·8%
GM 3+, 1−, 2−FF94·5 ± 4·2%0·0001
GM 17+, 1+, 2+FF27·8 ± 2·1%

Inhibition of the binding of A431/cetuximab complex to NK cells by GM 17+, 1+, 2+ and GM 3+, 1−, 2− allotypes of IgG1

To determine whether or not the GM allotype-associated differential inhibition of trastuzumab-mediated ADCC of SKBR-3 cells is specific to the cell line and the monoclonal antibody used, we repeated these experiments with cetuximab and HER1 over-expressing A431 cells. The results were comparable (Table 2). As with trastuzumab, IgG1 expressing the GM 3+, 1−, 2− allotypes was equally (albeit not as strongly) effective in inhibiting cetuximab-mediated ADCC of A431 cells, in the presence of NK cells expressing either V or F allele of FcγRIIIa (79·4 ± 5·5% versus 76·5 ± 2·4%; P = 0·47). In contrast, and similar to the results obtained with trastuzumab, IgG1 expressing the allelic GM 17+, 1+, 2+ allotypes was significantly more effective in inhibiting the cetuximab-mediated ADCC of A431 cells when NK cells expressed the V, rather than the F, allele of FcγRIIIa (83·3 ± 2·6% versus 50·3 ± 2·9%; P = 0·0001). For both therapeutic antibodies, IgG1 expressing the GM 17+, 1+, 2+ allotypes and homozygosity for the FcγRIIIa F allele was the least potent combination for inhibiting monoclonal antibody-mediated ADCC of target cancer cells.

Table 2.  Inhibition of cetuximab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) of A431 cell by natural killer (NK) cells expressing different FcγRIIIa genotypes in the presence of allotypically disparate immunoglobulin (Ig)G1 proteins.
IgG1 phenotypeFcγRIIIa genotypeInhibition of ADCCP-value
GM 3+, 1−, 2−VV79·4 ± 5·5%0·47
FF76·5 ± 2·4%
GM 17+, 1+, 2+VV83·3 ± 2·6%0·0001
FF50·3 ± 2·9%
GM 3+, 1−, 2−VV79·4 ± 5·5%0·35
GM 17+, 1+, 2+VV83·3 ± 2·6%
GM 3+, 1−, 2−FF76·5 ± 2·4%0·0002
GM 17+, 1+, 2+FF50·3 ± 2·9%

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

The association of certain Fc (GM)-FcγRIIIa genotypic combinations with higher inhibition of NK-mediated ADCC observed here might be a reflection of higher affinity between Fc and FcγRIIIa molecules expressing these genotypes. In the presence of both FcγRIIIa-VV- and FcγRIIIa-FF-expressing NK cells, the inhibitory effect on trastuzumab-mediated ADCC was higher for the IgG1 expressing GM 3+, 1−, 2− allotypes than that for IgG1 expressing the GM 17+, 1+, 2+ allotypes. This appears to be consistent with the results of a recent investigation on the measurement of IgG-FcγRIIIa binding [17]. These authors reported that for both FcγRIIIa V- and F-expressing cells, IgG proteins of GM 3 allotype bound slightly but reproducibly better than those expressing the GM 1, 17 allotypes.

The results presented here have important implications for antibody-based therapy in cancer patients. Musolino et al. [3] have reported that homozygosity for the V allele of FcγRIIIa, which was correlated significantly with objective response rate and progression-free survival, was also associated with significantly higher level of trastuzumab-mediated ADCC. Our results are consistent with this finding, as the constant region of trastuzumab is similar to the GM 17+, 1+, 2+ phenotype, in that it carries the GM 17 allotype although it lacks the GM 1 and 2 determinants. (The GM 1 allotype, which is in almost absolute linkage disequilibrium with GM 17 in Caucasians, was engineered out and replaced with the non-GM 1 isoallotype, to make trastuzumab less immunogenic [18].) The results reported by Musolino et al. imply that majority of people who carry the more common F allele [19] are unlikely to respond adequately to this therapy. We found that IgG1 proteins expressing GM 3+, 1−, 2− allotypes had high inhibitory effect on ADCC in the presence of both VV- and FF-expressing NK cells. This suggests that a HER2-targeting humanized monoclonal antibody, which expresses the GM 3+, 1−, 2− allotypes in the constant region, would be beneficial to the entire patient population, and especially to those (F-carriers) who are unlikely to respond adequately to the current trastuzumab therapy.

Monoclonal therapeutic antibodies with engineered/mutated Fc variants have shown dramatically higher ADCC in vitro[6]; however, as mentioned above, any harmful in vivo consequences of increased immunogenicity of these antibodies have not been investigated adequately. Therapeutic IgG antibodies with naturally occurring Fc variants (GM allotypes) are less likely to be immunogenic than those carrying the engineered variants. Therefore, to obtain the maximum clinical efficacy of humanized monoclonal antibodies, further studies are needed to explore the role of GM and FcγR loci in ADCC. Genes do not act in isolation: there is a growing body of evidence that epistasis – modification of the action of a gene by one or more other genes – plays a significant role in many structural and regulatory pathways [20]. Determinants expressed on Fc and FcγR are probably some of the most likely ligand-receptor candidate pairs for gene–gene interactions in the human genome, and based on other allelic ligand-receptor interactions involving genes of the immune system [21], it is reasonable to expect that such interactions contribute to the immunological pathways leading to the ADCC of tumour cells.

To our knowledge, this is the first report presenting evidence for the involvement of GM allotypes in ADCC.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

We thank the volunteers for their blood donation and Dr Twal Waleed for technical assistance with the Bio-Rad icycler. This work was supported in part by grants from the US Department of Defense (W81XWH-08-1-0373 and W81XWH-09-1-0329).

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  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References
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