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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

One of the major issues in current antibody therapy is insufficient efficacy. Various biological factors relating to the host’s immune system or tumor cells have been suggested to reduce the efficacy of anti-CD20 therapy in B-cell malignancies. In this study, we characterized the in vitro anti-lymphoma activity of anti-CD20 antibodies having a novel engineered heavy chain with enhanced complement-dependent cytotoxicity (CDC). Anti-CD20 antibodies having a variant heavy constant region of mixed IgG1/IgG3 isotype, which have previously been found to enhance CDC, were investigated for their in vitro CDC against lymphoma cells and whole blood B-cell depletion activity. Use of the variant constant region greatly increased the CDC of an anti-CD20 antibody having variable regions identical to those of rituximab to the level shown by an IgG1 antibody of ofatumumab. Although the whole blood assay showed different cytotoxicity patterns among individual blood donors, the CDC-enhancing variant of rituximab showed higher activity than the parent IgG1 and consistently showed maximized activity when further combined with antibody-dependent cellular cytotoxicity (ADCC)-enhancing modification by fucose removal from Fc-linked oligosaccharides. In addition, the rituximab variant showed potent CDC against transfectant cells with lower CD20 expression and chronic lymphocytic leukemia–derived cell lines with higher complement regulatory proteins. These findings suggest that CDC enhancement, both alone and in combination with ADCC enhancement, increases the anti-lymphoma activity of anti-CD20 antibodies irrespective of individual differences in effector functions, and renders current anti-CD20 therapy capable of overcoming the potential resistance mechanisms. (Cancer Sci 2009; 100: 2411–2418)

Although therapeutic antibodies now constitute a major class of novel therapeutic agents, current cancer immunotherapy still requires improvements in efficacy. Rituximab,(1) an anti-CD20 chimeric monoclonal antibody for the treatment of non-Hodgkin’s lymphoma, is one of the most successful examples of approved antibodies but is used in combination with chemotherapy because of its insufficient efficacy when given as monotherapy.(2) In addition, tumor cells remaining after rituximab treatment tend to be highly resistant to rituximab.(3)

Recent studies have suggested that the effector functions of antibodies play important roles in antibody therapy,(4–7) especially the effector functions that depend on Fcγ receptor (FcγR) IIIa,(8–10) which is present on natural killer (NK) cells, monocytes/macrophages, and dendritic cells. Of these, FcγRIIIa on NK cells is the major receptor triggering antibody-dependent cellular cytotoxicity (ADCC), but the involvement of this receptor in other cells of myeloid origin is not fully understood. Therefore, extensive efforts have been made to enhance ADCC by improving FcγRIIIa binding affinity by introducing amino acid mutations into the immunoglobulin Fc fragment (Fc)(11,12) or by modification of oligosaccharides linked to Asn297 in the Fc.(13–15)

In particular, removal of the fucose residue from the Fc-linked oligosaccharides critically affects ADCC.(14) In mammals, fucose residues are attached to the innermost N-accetylglucosamine residue of almost all Fc-linked oligosaccharides via an α-1,6 linkage,(16) and conventional antibodies produced with wild-type CHO cells possess almost fully fucosylated oligosaccharides and exhibit only modest ADCC.(14–17) We have generated an α-1,6-fucosyltransferase gene (FUT8) knockout CHO cell line (CHO/FUT8−/−) which can stably produce non-fucosylated antibodies,(18) and demonstrated that antibodies which are deficient in fucose residues of Fc-linked oligosaccharides show significantly augmented FcγRIIIa binding,(19) ADCC,(15) and antitumor activity in vivo.(20) ADCC enhancement by this method is also applicable to antibodies of other IgG isotypes(21) and antibody-like binding molecules having an Fc region.(22–24)

Antibody effector function also includes complement-dependent cytotoxicity (CDC), which is triggered by the binding of complement C1q subunits to the Fc regions of antibodies bound to cell surface antigens, and is followed by the cascadic activation of a series of complement proteins and resultant cytolysis. Recent studies have suggested that CDC is involved in the mechanisms underlying rituximab therapy.(3–5,25–28) Also, the importance of CDC activity in immunotherapy may be illustrated by the good clinical response of another anti-CD20 antibody, ofatumumab, which can induce much more potent CDC than rituximab as a consequence of its distinct epitope.(29) Fifty percent of patients achieved a clinical response in a recent phase I/II study of ofatumumab in chronic lymphocytic leukemia (CLL), which is highly resistant to rituximab therapy with a typical response rate of 0–30%.(30)

In order to improve the CDC activity of therapeutic antibodies, various approaches have been reported and there are several successful examples. The use of IgM antibodies or a mixture of multiple monoclonal antibodies including polyclonal antibodies(31) are possible means of improving CDC, because these methods increase the accessibility of C1q by the clustered Fc moiety, but require more validation and confirmation in terms of stable production and potential toxicity. In addition, artificially engineered heavy chain variants with amino acid mutations possessing improved C1q binding and enhanced CDC have been reported.(32,33) Recently, we reported a unique approach to the generation of CDC-enhancing constant region variants.(34) In this method, the Fc portion of the human IgG1 heavy chain is converted into IgG3 to create a novel chimeric constant region of mixed IgG1/IgG3 isotype, which possesses enhanced C1q binding and CDC.

Clinical tumors have various properties that hinder the efficacy of therapeutic antibodies, such as heterogeneous and/or decreased antigen expression in tumor cells(28) and increased expression of complement regulatory proteins (CRP).(3,25,28,35) In addition to tumor factors, various host factors (patient factors) have been suggested that might obscure the clinical response of therapeutic antibodies based on antibody effector functions: individual heterogeneity in ADCC,(8–10) variable NK cell number and different expression of functional receptors in NK cells among patients,(36,37) and down-regulation of ζ chains in effector cells.(38) Thus, the simultaneous enhancement of multiple effector functions, not restricted to the improvement of ADCC, may be a promising approach whereby enhanced multiple effector functions could compensate for each other.

We describe here how the enhancement of CDC by using variant constant regions potentiates the antitumor cytotoxic activity of anti-CD20 antibodies in terms of overcoming low antigen expression and high expression of CRPs in tumor cells and stable target killing using effectors derived from different individuals.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Cells and antibodies.  CHO/DG44, a wild-type CHO cell line,(39) was kindly provided by Dr Lawrence Chasin (Columbia University, New York, NY, USA). CHO/FUT8−/−, α-1,6-fucosyltransferase gene (FUT8) knockout CHO cell line for the production of IgG without fucosylation, was described previously.(18) CD20-positive chronic B-cell leukemia (B-CLL)-derived cell lines, EHEB, MEC-1, and MEC-2 were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). CD20-positive B lymphoma Raji, ST486, and Daudi cells were purchased from ATCC (Manassas, VA, USA). CD20/EL4 cell lines were established by human CD20 transfection into mouse thymoma EL4 cells and their CD20 expression was determined.(40) The production and purification of anti-CD20 antibodies have been reported.(34)

Blood donors.  Blood donors were randomly selected from healthy volunteers. All donors gave written informed consent before analyses in accordance with the process approved by the Institutional Ethics Committee in Kyowa Hakko Kirin Co. Ltd.

Expression analysis by flow cytometry.  To determine the absolute number of antibody-binding sites per cell, quantitative flow cytometric analysis was performed using Dako Qifikit (DakoCytomation, Kyoto, Japan) for CD20 antigen and CRPs. Briefly, 2 × 105 cells were incubated in the presence of a saturating concentration (100 μg/mL) of antigen-specific mouse IgG1 antibody (anti-CD20 clone MEM-97 [Exbio, Prague, Czech Republic], anti-CD46 clone MEM-258 [Exbio], anti-CD55 clone 67 [Abcam, Tokyo, Japan], anti-CD59 clone BRA-10G [Thermo Scientific, Fremont, CA, USA], isotype control clone MOPC-21 [BioLegend, San Diego, CA, USA]), and then washed and stained with FITC-conjugated antimouse IgG (DakoCytomation) on ice. Standard beads coated with a known amount of mouse IgG molecules were also stained with FITC-conjugated anti-IgG. The stained samples were analyzed using a flow cytometer (Cytomics FC 500; Beckman Coulter, Tokyo, Japan), and the number of binding sites per cell was calculated by comparing the median fluorescence intensity value of the cells to a standard curve calculated from the fluorescence intensity values of standard beads.

ADCC assay.  PBMCs were prepared from blood using Lymphoprep (Axis Shield, Dundee, UK). Target cells (1 × 104) were incubated with anti-CD20 antibody and human PBMCs as effector cells at an effector/target cell ratio of 20:1 for 4 h at 37°C. After incubation, lysed target cells were detected by a lactate dehydrogenase release assay and the percent cytotoxicity was calculated as described previously.(18)

CDC assay.  CDC assays were performed as described previously.(33) Briefly, target cells (5 × 104) were incubated with various concentrations of anti-CD20 antibody and human serum (Sigma, St. Louis, MO, USA) as the source of complement at a dilution of 1/6 in supplemented RPMI-1640 for 2 h at 37°C in 96-well flat-bottomed plates. In some experiments, 25 μg/mL of anti-CRP antibodies were added. After incubation, the cell proliferation reagent WST-1 (Roche Diagnostic, Basel, Switzerland) was added (15 μL/well) and the plates were further incubated for 4 h to detect the live cells. The absorbance (A450–A650) of the formazan dye produced by metabolically active cells of each well was detected on an Emax plate reader (Molecular Devices, Tokyo, Japan). Cytotoxicity was calculated according to the formula:

  • image

where E is the absorbance of experimental well, S is that in the absence of mAb (cells were incubated with medium and complement alone), and M is that of medium and complement in the absence of target cells and antibody.

Whole blood assay.  Peripheral blood from healthy donors was incubated with anti-CD20 antibody, and the depletion of B cells was detected by flow cytometry. In 24-well flat-bottomed plates, 500 μL of peripheral blood and 100 μL of RPMI-1640 medium containing serial dilutions of anti-CD20 antibody were mixed and incubated for 4 h at 37°C. After incubation, aliquots of samples were stained with PE/Cy5-anti-CD2 (BioLegend) and FITC–anti-CD19 (BioLegend) for 30 min at 4°C. Mouse IgG1 (clone MOPC-21) labeled with PE/Cy5 or FITC (BioLegend) was used as isotype control. Stained cells were suspended in FACS lysing solution (BD Biosciences, Tokyo, Japan) to lyse erythrocytes, washed twice with 1% BSA-PBS, and analyzed on a flow cytometer. Isotype controls were used to determine the cut-off points for CD19+ and CD2+ cells. The percentage of CD19+ CD2 B cells was calculated according to the following formula:

  • image

where E is the number of CD19+ CD2 B cells in the experimental well treated with anti-CD20 antibodies, and M is that in the medium-treated well.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Enhancement of CDC activity of anti-CD20 antibodies by using variant constant regions of mixed IgG1/IgG3 isotype.  We have recently demonstrated that engineered heavy chain constant regions, constructed as mixtures of human IgG1 and human IgG3 isotypes, showed enhanced CDC activity that exceeded the level achieved by either wild-type IgG1 or IgG3.(34) The purpose of the present study was to characterize anti-lymphoma potential of CD20 antibodies armed with a variant constant region 113F, one of the potent CDC-enhancing variants screened from a panel of mixed IgG1/IgG3 sequences. In the amino acid sequence of 113F, the Fc region, except a small portion of the C-terminus, was identical to that of IgG3, while other regions were those from IgG1.

First, we constructed two sets of anti-CD20 antibody clones, each including both wild-type IgG1 and 113F-type variants, whereas the light chains were fixed as the κ isotype. For the V region sequences, those of the two clinically proven anti-CD20 antibodies, rituximab (clone 2B8) and ofatumumab (clone 2F2), were employed. We compared the CDC of the four anti-CD20 antibody variants (Fig. 1), that is 2B8-IgG1, 2B8-113F, 2F2-IgG1, and 2F2-113F, against two human lymphoma cell lines, Raji cells (Burkitt’s lymphoma) and EHEB cells (chronic lymphocytic leukemia). In the comparison between the two wild-type IgG1 antibodies, 2F2-IgG1 showed much more potent CDC activity than 2B8-IgG1, which is in agreement with a previous in vitro study of ofatumumab.(41) In addition, the use of the variant constant region showed significant enhancement of CDC for both anti-CD20 clones. Importantly, 2B8-113F showed a very similar level of CDC to that of 2F2-IgG1, implying that the degree of CDC enhancement conferred by the variant constant regions may be of therapeutic value, as evidenced by the better outcomes of the use of ofatumumab in CLL patients compared with rituximab therapy.(30)

image

Figure 1.  Anti-CD20 antibodies with IgG1/3 mixed isotype heavy chain exert enhanced lymphoma cell killing. Complement-dependent cytotoxicity (CDC) activity against B lymphoma cell line Raji (A) and chronic lymphocytic leukemia (CLL) cell line EHEB (B) are shown as mean ± SD of triplicates. Symbols represent 2B8-IgG1 (○), 2B8-113F (•), 2F2-IgG1 (Δ), and 2F2-113F (bsl00066).

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Enhanced cytotoxic activity in human blood.  It has been demonstrated previously that variant 113F lacking fucosylation in Fc-linked oligosaccharides exerts enhanced ADCC and CDC.(34) To examine the cytotoxic activity of anti-CD20 antibodies with dual-enhanced effector functions in more clinically relevant settings, we performed a whole blood assay.(42,43) In this assay system, anti-CD20 antibodies can induce ADCC and CDC against B cells simultaneously by utilizing NK cells and serum complements as an effector source. Prior to the assay, we compared ADCC and CDC activities using PBMCs and serum separated from the same blood sample of a single donor as effectors, respectively, and found that ADCC and CDC were induced at different antibody concentration ranges; CDC was induced at 1000-fold higher concentration than CDC for ADCC in this case (Fig. 2).

image

Figure 2.  Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) using effectors from a single blood donor. ADCC (A) and CDC (B) of anti-CD20 antibody variants against chronic lymphocytic leukemia (CLL) cell line MEC-1 were measured using PBMCs and serum separated from the same blood sample of a single donor as effectors, respectively. Symbols represent 2B8-IgG1 (○), 2B8-IgG1-nf (•), 2B8-113F (Δ), 2B8-113F-nf (bsl00066), and 2B8-IgG4 (×). The mean ± SD of triplicates are shown.

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B cell-depleting activity was then determined using a whole blood assay in which anti-CD20 antibodies were coincubated with peripheral blood from healthy volunteers and then the percentage of CD20-positive viable B cells remaining was determined by flow cytometric analysis. Five variants of anti-CD20 antibodies were tested for this activity (Fig. 3): 2B8-IgG1 (IgG1-fucosylated variant), 2B8-IgG1-nf (IgG1-nonfucosylated variant with enhanced ADCC), 2B8-113F (113F-fucosylated variant with enhanced CDC), 2B8-113F-nf (113F-nonfucosylated variant with enhanced CDC and ADCC), and 2B8-IgG4 (IgG4-fucosylated variant with low ADCC and low CDC). In blood from donor 1, both CDC-enhancing variant 2B8-113F and ADCC-enhancing variant 2B8-IgG1-nf depleted more B cells than the parent 2B8-IgG1. The enhancement in B-cell depletion was more pronounced for the ADCC-enhancing variant 2B8-IgG1-nf than for the CDC-enhancing variant 2B8-113F, especially at lower concentrations. In this case, the dual-enhancing variant 2B8-113F-nf showed similar activity to the ADCC-enhancing variant. On the other hand, in blood from donor 2, B cells were significantly decreased neither by the parent IgG1 nor by the ADCC-enhancing variant, whereas the dual-enhancing and CDC-enhancing variants exerted marked activities, suggesting that enhancing CDC activity seemed more effective in eliminating B cells in this case. In donor 3, improvement of B-cell depletion from IgG1 was smaller than for other blood samples, and all the enhancing variants showed similar activities. Taken together, these results indicate that although the enhancement of either ADCC or CDC individually contributed to improved target cell killing by anti-CD20 in the whole blood assay, the simultaneous enhancement of both effector functions constantly led to maximal killing of B cells irrespective of the individual difference in responsiveness to each effector function.

image

Figure 3.  B-cell depletion activity of anti-CD20 antibody variants in whole blood assay. Anti-CD20 antibodies were added to whole blood from three donors (A–C), and remaining CD20+ B cells were measured by flow cytometer after incubation. Percentage of CD2+ CD19 T cells was not changed by anti-CD20 antibodies (data not shown). Symbols represent 2B8-IgG1 (○), 2B8-IgG1-nf (•), 2B8-113F (Δ), 2B8-113F-nf (bsl00066), and 2B8-IgG4 (×). The mean ± SD of triplicates are shown.

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CDC activity on cells expressing fewer CD20 molecules.  Next, we investigated the impact of CD20 density on the CDC of rituximab and its CDC-enhancing variant, since inter- or intra-patient heterogeneity in the expression of antigen was suggested as a mechanism of resistance in anti-CD20 therapy.(44) With this aim, we examined the CDC activity of anti-CD20 (2B8) variants against transfectant cells expressing variable numbers of CD20 molecules (1.2 × 104 to 5.8 × 105 CD20 molecules/cell).(40) We found that the variant 113F exerted higher CDC than parent IgG1 in all transfectants (Fig. 4A), and the analysis at a fixed concentration of mAb indicated that the effective range of CD20 expression for CDC was one order lower for variant 113F compared with parent IgG1 (Fig. 4C). Notably, variant 113F showed significant CDC activity even against targets with lower CD20, whereas parent IgG1 showed only marginal activity (Fig. 4A upper left).

image

Figure 4.  Complement-dependent cytotoxicity (CDC) of anti-CD20 antibody variants against low CD20-expressing cells. (A) CDC activity against human CD20 transfectant EL4 cells expressing 1.2 × 104/cell (upper left), 4.1 × 104/cell (lower left), 6.7 × 104/cell (upper right), and 2.1 × 105/cell (lower right). (B) Anti-CD20 antibodies showed no CDC activity against parent murine EL4 cells. (C) CDC activity at a fixed concentration (1.1 μg/mL) of 2B8-IgG1 (•), 2B8-113F (bsl00066), and 2B8-IgG4 (×) against transfectant cells expressing variable numbers of CD20 molecules. The mean ± SD of triplicates are shown.

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CDC activity on lymphoma cell lines highly resistant to rituximab CDC.  Next, we further explored the impact of enhancing CDC on the killing of natural lymphoma cell lines, especially for those that respond poorly to rituximab CDC. For this aim, we selected three cell lines (EHEB, MEC1, and MEC2) derived from CLL, which is known to be resistant to rituximab therapy compared with other susceptible non-Hodgkin's lymphoma (NHL) subtypes,(41,45) and compared the CDC activity of the anti-CD20 (2B8) variants against these cell lines (Fig. 5). (Note that these cell lines were not truly CLL cells, but probably derived from non-malignant cells that underwent prolymphocytic transformation during disease progression, because a true CLL cell line is difficult to establish due to its intrinsic resistance to immortalization.)(46–48) 113F variants with different fucosylation showed strong killing activity in all cell lines examined, whereas IgG1 showed only marginal cytotoxicity in the selected CLL-derived cell lines (Fig. 5A–C) except for a relatively sensitive Burkitt’s cell line Daudi (Fig. 5D). As demonstrated previously,(21,34) the presence or absence of fucose in Fc-linked oligosaccharides did not seem to affect CDC. These results indicate that the use of mixed isotype heavy chains significantly improves the CDC activity of rituximab against CDC resistant cells.

image

Figure 5.  Complement-dependent cytotoxicity (CDC) of anti-CD20 antibody variants in chronic lymphocytic leukemia (CLL) cells. CDC activity against CLL cell lines EHEB (A), MEC-1 (B), MEC-2 (C), and B lymphoma line Daudi (D) are shown. Symbols represent 2B8-IgG1 (○), 2B8-IgG1-nf (•), 2B8-113F (Δ), and 2B8-113F-nf (bsl00066). The mean ± SD of triplicates are shown.

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CRPs as key factors that protect cells from rituximab CDC.  CRPs, such as CD46 (membrane cofactor protein), CD55 (decay-accelerating factor), and CD59 (protectin), protect cells from complement activation. Several reports have suggested the involvement of CRPs in the rituximab resistance mechanisms of CLL.3,28 We determined the expression level of CRPs in lymphoma cell lines, including the three CLL-derived cell lines resistant to rituximab CDC, by quantitative flow cytometry, and confirmed that the resistant cells lines, in comparison with Burkitt’s lymphoma cells lines, expressed higher level of CRPs, especially CD59 (Table 1). On the other hand, the resistant cell lines expressed a level of CD20 comparable to that of the Burkitt’s lymphoma cell lines, suggesting that the resistance mechanism of these cell lines involves high expression of CRPs. Further supporting this, addition of neutralizing anti-CRP antibodies canceled the CDC resistance of the three cell lines, and this effect was more pronounced for IgG1 than for 113F (Fig. 6).

Table 1.   Expression level of CRPs in lymphoma cell lines
Cell lineExpression (×104 binding sites/cell)Cancer type
CD20CD46CD55CD59
Daudi21.45.61.40.5Burkitt’s lymphoma
ST48626.83.81.02.6
Raji25.94.82.85.0
MEC-132.45.83.213.3Chronic lymphocytic leukemia
EHEB68.95.70.517.2
MEC-258.010.75.536.0
BALL-122.17.52.92.8Acute lymphocytic leukemia
NCI-N4170.13.20.113.9Small cell lung carcinoma
image

Figure 6.  Effect of complement regulatory proteins (CRP) neutralization on complement-dependent cytotoxicity (CDC) sensitivity of chronic lymphocytic leukemia (CLL) cells. CDC activity of anti-CD20 antibodies against CLL cell line EHEB was measured in the absence or the presence of CRP neutralizing antibodies. Symbols represent 2B8-IgG1 alone (○) 2B8 plus anti-CD46 (□), 2B8 plus anti-CD55 (◊), 2B8 plus anti-CD59 (Δ), 2B8 plus mixture of 3 anti-CRPs (×), 2B8-113F alone (•), 2B8-113F plus anti-CD46 (bsl00001), 2B8-113F plus anti-CD55 (◆), 2B8-113F plus anti-CD59 (bsl00066), 2B8-113F plus mixture of three anti-CRPs (+). Anti-CRP antibodies were added at 25 μg/mL, except for the experiments with a mixture of three anti-CRPs (8.3 μg/mL each). The mean ± SD of triplicates are shown. In the absence of anti-CD20 antibody, anti-CRP antibodies did not exhibit any cytotoxicity (data not shown).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

In antibody therapy, important roles of effector functions, depending in particular on FcγRIIIa, which is the major receptor triggering ADCC, have been suggested by several studies.(4–10) Therefore, enhancing ADCC by improving FcγRIIIa-binding capacity by the use of variant constant regions, including engineered Fc sequences and modification of oligosaccharides in the Fc region, would be of significant therapeutic value.

However, clinical tumor cells potentially avoid ADCC attack from therapeutic antibodies by various mechanisms, such as insufficient recruitment of effector cells due to low penetration into tumor tissues, myelosuppression of effector cells by toxic chemotherapy, loss or decrease of antigen in tumor cells, individual heterogeneity of ADCC (due to variation in NK cell number and functional polymorphism of FcγRIIIa), functional impairment of NK cells in cancer patients (such as down-regulation of the ζ chain),(38) and the inhibitory effect of serum IgG on ADCC.(43)

One possible solution for these potential issues is to maximize ADCC itself to levels that can overcome these inhibitory factors. We have previously demonstrated that nonfucosylated antibodies can mediate potent ADCC activity even for effector cells from individuals with weak ADCC,(19) for target cells with lower antigen expression,(40) or in the presence of serum IgG.(43) Alternatively, additional enhancement of other antibody functions (e.g. CDC, phagocytosis, and tumor penetration), and not relying on enhancing ADCC alone, may add to conventional ADCC-based therapeutics.

Recently, important roles of CDC in antibody therapy were highlighted by the different clinical responsiveness in CLL patients of the two different anti-CD20 antibodies rituximab and ofatumumab, even if it is difficult to directly compare the different phase studies.(30,49–52)

Generally, CDC activation requires a higher concentration of antibodies than ADCC, which was also confirmed in this study (Fig. 2). Enhanced ADCC would be especially beneficial at lower concentrations, which was underscored in a whole blood assay (Fig. 3, donor 1). However, even more important is that the dual-enhancing antibody (variant 2B8-113F-nf) constantly showed the best activity among all variant antibodies, whereas enhancing either ADCC or CDC alone did not always lead to maximal cytotoxicity for different blood donors, probably because of individual heterogeneity in each effector function.

Regarding methods of enhancing antibody effector functions by introducing amino acid mutations into the Fc region, undesired influences on other functions have often been reported, such as the decrease in FcγRIIIa-binding capacity after introducing amino acid mutations that improve binding to C1q,(32) FcγRIIa,(53) and neonatal Fc receptors.(54) In contrast, the 113F-type constant region showed no alteration in ADCC, and removal of fucose from 113F maximized both FcγRIIIa binding and ADCC to a similar extent as wild-type IgG1 antibodies.(34)

The reason why this variant sequence composed of a mixed IgG1/IgG3 isotype does not have an unpredicted influence on ADCC remains elusive, and it would be interesting to know the crystal structure of this molecule. In turn, because of the structure of nonfucosylated antibodies, removal of the fucose residue does not alter the overall crystal structure of the IgG1 Fc region except for the fine change in the hydration mode around Asn297.(55) The fact that the structure of IgG is not affected by removal of fucose means that this method is applicable to a wide variety of antibody species without influencing other intrinsic properties, as evidenced by successful ADCC augmentation in other IgG subclasses,(21) Fc fusion proteins,(22–24) and 113F-type antibodies.

Another interesting feature of CDC-enhancing anti-CD20 antibodies is that they can induce CDC activity in target cells with lower antigen expression, as revealed by the analyses using experimental transfectant cells as targets (Fig. 4). This might be of potential clinical benefit, since some B-cell-derived malignancies having low CD20 expression showed a relatively poor response to rituximab, such as small lymphocytic leukemia(26) and CLL,(56) although other resistant mechanisms are possible in these cancer types. In addition, it has been suggested that the expression level of CD20 molecules may vary over a broad range within a patient(57) so that a low CD20 population may persist after rituximab therapy.

In another set of experiments (Fig. 5), we examined CDC activity against natural CLL-derived cell lines with high resistance to rituximab CDC. In agreement with the poor responsiveness of this disease to rituximab therapy, the present results also demonstrate that the chosen cell lines are highly resistant to the CDC of rituximab. However, the CD20 expression of these lines was similar to, or even higher than, that of other lymphomas (Fig. 6), in contrast to a report in which tumor cells from CLL patients showed lower expression of CD20,(28) probably due to their possible origins of prolymphocytic transformation from non-malignant cells. Instead, these cell lines express high levels of CRPs, which protect cells from CDC by inhibiting the complement protein activation cascade. The most prominent up-regulation was found for CD59, which is known to correlate with rituximab CDC and was found on persistent rituximab-bound cells in patients(25) or may be up-regulated after rituximab therapy.(3) Although the predictive value of CRPs remains controversial (no relationship has been found between CRP expression and the response to rituximab),(58) CRPs do inhibit CDC by rituximab against clinical CLL samples,(28) and complement sensitivity in vitro is augmented by neutralizing CRPs.(59–63) Interestingly, CDC mediated by the 113F variant showed higher CDC than IgG1 even with the neutralization of CRPs, suggesting that the use of the 113F-variant heavy chain may overcome the potential resistance mechanisms involving CRPs; up-regulated activation of complement proteins on target cells by the CDC-enhancing variant might exceed the protective capacity of CRPs.

A major obstacle to predicting the clinical effectiveness of engineered antibodies with enhanced effector functions might be the lack of an appropriate animal tumor model. It is generally accepted that the immune systems of animals are far different from that in humans in terms of effector functions; for example, the most widely used murine tumor models do not have the capacity to induce ADCC or CDC.(62) However, importantly, good antitumor activity seen in the early stage clinical trials of ofatumumab may provide a clue to this unanswered question; enhancing complement activation may add to the therapeutic efficacy of approved antibodies.

In conclusion, we demonstrate here the potential therapeutic value of variant anti-CD20 antibodies with enhanced CDC activity by using engineered constant regions of mixed IgG1/IgG3 isotype. Furthermore, the combination of this CDC-enhancing modification with the ADCC-enhancing removal of fucose may be a promising way of overcoming the diverse mechanisms whereby cells resist antibody therapy.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
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