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. 2005 Apr 22;54(12):1172–1179. doi: 10.1007/s00262-005-0686-1

A DeImmunized chimeric anti-C3b/iC3b monoclonal antibody enhances rituximab-mediated killing in NHL and CLL cells via complement activation

Wu Peng 1,1,✉, Xin Zhang 1, Nehal Mohamed 1, Giorgio Inghirami 2, Kenichi Takeshita 3, Andrew Pecora 4, Linda L Nardone 1, Steve E Pincus 1, Leslie S Casey 1, George L Spitalny 1
PMCID: PMC11034342  PMID: 15846490

Abstract

Complement-dependent cytotoxicity (CDC) is a key mechanism of Rituximab (RTX) action in killing non-Hodgkin’s lymphoma (NHL) cells both in vitro and probably in vivo. A DeImmunized, mouse/human chimeric monoclonal antibody (Mab), H17, specific for cell-associated complement C3 cleavage products, C3b and iC3b, was generated to enhance RTX-mediated killing of target cells by CDC. When NHL cell lines were treated with RTX and H17 in the presence of complement for 1 h, there was 40–70% more cell death than that observed with RTX alone. The enhancing effect of H17 was also seen over longer treatment periods. H17 was tested ex vivo against primary cells from NHL and chronic lymphocytic leukemia (CLL) patients. In RTX-resistant NHL samples, H17 enhanced RTX-mediated killing; in the remaining samples RTX + complement alone promoted more than 80% killing, and no significant enhancement was observed. The H17 antibody also increased RTX-mediated killing in four out of nine CLL samples. H17 may have therapeutic applications in NHL and CLL treatment as an adjunctive therapy to RTX. It might also enhance the activity of other therapeutic antibodies that work through CDC.

Keywords: Complement, Rituximab, Antibody therapy, Non-Hodgkin’s lymphoma, Chronic lymphocytic leukemia

Introduction

The use of therapeutic monoclonal antibodies (Mabs) is a promising approach in the treatment of hematologic malignancies, since a number of antibodies offer efficient and specific cell killing with minimal side effects [1, 13, 30, 33].

Rituximab (RTX) is a chimeric human IgG1 anti-CD20 Mab that is approved by the FDA for the treatment of relapsed or refractory indolent non-Hodgkin’s lymphomas (NHL). The overall response (OR) rate is 48%, with a complete response (CR) rate of 6% [26]. Currently RTX is in clinical trials as a single-agent first-line and maintenance treatment or, in combination with chemotherapy for indolent and follicular NHL or aggressive NHL. Also, RTX is being tested for mantle cell lymphoma, chronic lymphocytic leukemia (CLL), and autoimmune diseases (see Boye et al. [1] and references therein). In a phase II trial using RTX as first-line and maintenance treatment for indolent NHL, the OR (73%) and CR (37%) were higher than for refractory indolent NHL [14]. However, there is still a need for improvement, which may be achieved by combining RTX with other therapeutic modalities [17].

Complement-dependent cytotoxicity (CDC) is now viewed as one of the major mechanisms by which RTX kills targets in vitro, and probably in vivo [4–6, 16, 22, 25]. Lymphoma cells often over-express complement regulatory proteins (CRPs) that interfere with C3b deposition and membrane attack complex (MAC) formation [11, 12, 32], making them resistant to CDC. Cells can also become CDC-resistant by developing reduced susceptibilities to lysis by MAC, for example through alterations in membrane properties that allow recovery from what would otherwise be lethal damage [21]. RTX activates complement upon binding to CD20 on B cells, resulting in deposition of C3 cleavage products, C3b and iC3b [collectively called C3b(i)], on the cells [22, 23]. If complement-activating antibodies can recognize cell-associated C3b(i), they may enhance CDC mediated by Mabs such as RTX.

A murine Mab, 3E7, against C3b(i) was found to act as an adjuvant to RTX [22]. In the presence of RTX and normal human serum (NHS) as a source of complement in vitro, 3E7 preferentially bound to cell-associated C3b(i) generated during complement activation by RTX. This binding triggered further complement activation and enhanced CDC in NHL cell lines. The ability of 3E7 to bind to cell-bound C3b(i) was also demonstrated in a non-human primate model [22]. These results suggested the potential for 3E7 to be developed into a therapeutic Mab.

Here, we present the re-engineering of murine anti-C3b(i) Mab 3E7 to produce a chimeric DeImmunized human IgG1. In this antibody, certain DNA sequences in the mouse variable region that encode amino acid sequences predicted to be immunogenic in humans are mutated to encode non-immunogenic sequences (DeImmunized). The DeImmunized, chimeric anti-C3b(i) therapeutic Mab candidate, H17, enhances RTX-mediated killing of NHL cell lines as well as primary NHL and CLL cells. The results support further clinical development of H17 as adjunctive therapy with RTX for treatment of NHL and CLL.

Materials and methods

Cell cultures and primary cells

Raji and DB NHL cell lines were obtained from ATCC (Manassas, VA, USA). Rituxan-resistant Raji cell line (Raji-R) was the gift of Genmab (Utrecht, The Netherlands). All cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT, USA). Bone marrow aspirates from NHL patients were obtained from Hackensack University Medical Center (Hackensack, NJ, USA). Frozen lymph node cell suspensions from NHL and CLL patients were obtained from New York University (New York, NY, USA). All patient samples were obtained under IRB-approved protocols. For bone marrow samples, a consent form was also obtained from each patient.

Antibodies and reagents

Rituxan (IDEC, chimeric anti-human CD20, Rituximab) was purchased from a hospital pharmacy. The hybridoma cell lines producing anti-C3b(i) Mabs 3E7 and 1H8 [24] were provided by Dr. Ronald Taylor (University of Virginia, Charlottesville, VA, USA). 3E7 and 1H8 Mabs were purified from mouse ascites fluid by protein A affinity chromatography. Biotin-labeled 3E7 and FITC-labeled 1H8 were prepared using kits from Pierce (Rockford, IL, USA). Chimeric 3E7 and DeImmunized anti-C3b(i) Mabs including H17 were purified by protein A chromatography from culture supernatant. All flow cytometry reagents were purchased from BD Biosciences (San Diego, CA, USA) or Jackson ImmunoResearch (West Grove, PA, USA), except that goat anti-human C1q and purified human iC3b and C3b were from Advanced Research Technology (San Diego, CA, USA).

Normal human serum was prepared from healthy donors or purchased from Sigma (St Louis, MO, USA). Ficoll-Paque Plus was purchased from Amersham Pharmacia (Piscataway, NJ, USA). Alamar Blue reagent was purchased from Biosource (Camarillo, CA, USA).

CDC assay

For most CDC experiments with cell lines, a procedure published previously [22] was followed with some modifications. Briefly, 105 cells were treated with RTX with or without anti-C3b(i) Mab in the presence of NHS as a source of complement for various times at 37°C in 5% CO2. For Raji cells, a final serum concentration of 5% was used; for all other cell lines, 25%. After two washes with PBS–1% BSA, cells (in 100 μl) were stained with 10 μl manufacturer-prediluted FITC-anti-CD45 at room temperature (RT) for 20 min, washed, and then incubated with 10 μl propidium iodide (PI) (50 μg/ml) at RT for 5 min. Data were acquired for 1 min on a FACSCalibur (BD Biosciences, San Diego, CA, USA) at a flow rate of 60 μl/min. Cells were gated first on forward scatter and side scatter, then on CD45 and PI staining. The CD45+ PI− population was regarded as live cells. Cell killing was calculated using the following formula: Killing (%)=100x [1 – (Live cell # in treated sample/Live cell # in untreated control)]. Unless otherwise indicated, a saturating concentration of 10 μg/ml for both RTX and H17 was used.

To measure killing in lymphocytes, red blood cells from whole blood were lysed with non-fixing lysis buffer (Roche, Indianapolis, IN, USA) and white blood cells were washed with PBS and recovered by centrifugation at 200g for 5 minutes. Cells (5×105) were then treated with RTX and H17 and 25% autologous serum for 1 h at 37°C, 5% CO2. After washing, cells were stained with FITC-anti-CD45, APC-anti-CD19 and PI, and analyzed by flow cytometry. B lymphocytes were identified based on CD45-side scatter and CD19 staining. T and NK cells were identified as the CD19 negative lymphocyte population.

For experiments with patient samples, mononuclear cells from fresh bone marrow aspirates from each NHL patient or frozen CLL cells were obtained by Ficoll-Paque separation. Cells (5×105) were then treated as above. Alternatively, frozen lymph node cell suspensions from NHL patients were thawed, and up to 5×105 cells were treated as above. The CD20 level was expressed in molecules of equivalent soluble fluorochrome (MESF) calculated from PE-labeled beads (BD). Since RTX only works on CD20+ B cells, the fraction of B cells (identified by CD19+) that expressed CD20 was determined in a separate experiment. PI negative B cells were treated as live B cells. Cell killing (%)=100 × [1-(live B cell # in treated sample/live B cell # in untreated control)]/fraction of CD20+ cells from B cells.

Alamar blue assay

After 105 Raji-R cells were treated in a CDC experiment for 1 h, the cells were washed once in PBS/1% BSA, then suspended in 1 ml of RPMI-1640 medium supplemented with 1% heat-inactivated fetal bovine serum. A 100 μl sample was transferred into a well on a 96-well flat-bottom cell culture plate (Cellstar, Greiner Bio-One Inc., Longwood, FL, USA), and then 10 μl of Alamar Blue reagent was added to each well according to the manufacturer’s instruction. Cells were incubated at 37°C, 5% CO2 overnight. Samples with known cell numbers were also incubated with Alamar Blue as standards. The fluorescence of the reduced Alamar Blue reagent by an enzyme in live cells was measured (Ex=530 nm, Em=580 nm). Live cell number in each sample was calculated against the standard curve, and cell killing (%) =100× [1 – (Live cell # in treated sample/Live cell # in untreated control)].

C1q binding and C3b(i) deposition

For determination of C1q and C3b(i) on cells, cells were treated in the same way as in the CDC assay, except the incubation time was 30 min. After washing, cells were stained with FITC-1H8 [an anti-C3b(i) Mab that does not compete with H17], or goat anti-C1q and FITC labeled secondary antibody. Geometric means of fluorescence intensity were obtained from the live cell population by flow cytometry.

Construction of chimeric and DeImmunized anti-C3b(i) Mabs

Vectors containing the genes of variable domains, VH and VL, of mouse Mab 3E7, after verification of the DNA sequences, were transferred into eukaryotic expression vectors containing human γ1 and κ constant regions, respectively. Those vectors were co-transfected into NS0 cells by electroporation and clones were screened for production of human IgG in culture supernatants via a human IgG1/κ ELISA. The highest producing clone was selected as the cell line to produce chimeric 3E7.

Utilizing its proprietary modeling technique “peptide threading”, Biovation (UK) determined which mouse variable region sequences had the potential to bind to human MHC class II and elicit an immune response. Some or all of such sequences in the mouse variable region were then mutated by overlapping PCR into non-immunogenic sequences (DeImmunization). The modified variable regions were then spliced to the human γ1 constant regions and the constructs were transfected into NS0 cells for expression of DeImmunized chimeric 3E7s.

Anti-C3b(i) competitive ELISA

Microtiter plates were coated with 100 μl of 1 μg/ml human IgM (Sigma) in sodium bicarbonate buffer at 4°C overnight, washed, and blocked with 250 μl/well SuperBlock (Pierce, Rockford, IL, USA) for 1 h at RT. After washing, 50 μl/well of 5% NHS was added and incubated for 1 h at 37°C to allow C3b(i) deposition. After washing, serially diluted anti-C3b(i) antibody (50 μl/well) was added and incubated as above. Biotinylated 3E7 was then added and incubated for another hour. After washing, HRP-Streptavidin (Jackson ImmunoResearch) was added, the plate was incubated for 1 h at 37°C, and then washed. 3,3′,5,5′-tetramethyl-benzidine (TMB) was used as the substrate for HRP. Absorbance was measured at 450 nm on a SPECTRAmax 340PC reader. Data were analyzed using SOFTmax PRO 4.0 (Molecular Devices Co.). The binding to C3b(i) as a function of antibody concentration was fit into a 4-parameter curve.

Affinity measurement

To determine the affinity of the Mabs for C3b, kinetic measurements were performed using a BiaCore 3000 (BiaCore Inc., Piscataway, NJ, USA). Purified C3b was linked to a CM5 chip via amine coupling. Anti-C3b(i) Mab was then injected at various concentrations. All Mabs were diluted in BiaRunning buffer (Biacore) containing 1 mg/ml of carboxy-methylated dextran (Carbomer Inc. Westborough, MA, USA) to minimize non-specific binding. After each cycle chips were regenerated using 3 M potassium isothiocyanate in 50 mM Tris base for 30 s at 50 μl/min. Sensograms were analyzed using BIAevaluation software (BiaCore) to yield the kinetic constants.

Results

Enhancement of RTX-mediated cell killing by murine anti-C3b(i) Mab 3E7

The ability of 3E7 to enhance RTX-mediated killing by CDC [22] was confirmed using NHL cell line Raji. When Raji cells were treated with RTX and 3E7 in the presence of complement for 1 h, there was 15–50% more cell death than that observed with RTX alone (data not shown). The percentage of cell killing by CDC relative to untreated controls matched previously published results for Raji cells [22]. Heat-inactivated human serum (56°C, 1 h) or EDTA-treated serum gave less than 10% killing of Raji cells in the presence of RTX with or without 3E7 (data not shown), supporting the hypothesis that cell death was mediated through complement.

Construction and selection of chimeric DeImmunized anti-C3b(i) Mab H17

To generate candidate therapeutic Mabs, a mouse variable/human constant region chimeric construct of 3E7 (IgG1κ) was generated, and subsequently DeImmunized to further reduce the potential immunogenicity in humans (see Materials and Methods). The human γ1 constant region was chosen because IgG1’s can activate complement, bind to Fc receptors, and are generally amenable to process development.

To make sure the chimerization process did not compromise chimeric 3E7 and to select a DeImmunized Mab for development, chimeric 3E7 and the panel of DeImmunized Mabs were evaluated in comparison to murine 3E7 based on (a) potency in the CDC assay, (b) affinity for C3b using a BiaCore 3000 system, and (c) C50 value in an anti-C3b ELISA. Based on the ranking in those 3 assays, and the number of amino acids altered in the variable region (to reduce potential immunogenicity) of the DeImmunized Mabs, clone H17 was selected for further studies. As shown in Fig. 1, the enhancement in RTX-mediated- cell killing of NHL cell lines Raji-R and DB by chimeric 3E7 and H17 was similar to that by murine 3E7. Isotype controls for murine 3E7, chimeric 3E7 and H17 were negative in enhancing RTX-mediated killing (data not shown). The dissociation constants for murine 3E7, chimeric 3E7 and H17 were 1.1, 2.5, and 1.4 nM, respectively. In the anti-C3b(i) competition ELISA, the concentration of Mab that was needed to reduce binding of biotinylated-3E7 to 50% of maximum was 83.0, 49.7, and 48.9 ng/ml for murine 3E7, chimeric 3E7 and H17, respectively. Those results confirmed that all the Mabs behaved similarly.

Fig. 1.

Fig. 1

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Comparison among murine 3E7, chimeric 3E7 (Ch3E7) and H17 in CDC assays. a Raji-R cells were treated with 10 μg/ml RTX and 10 μg/ml murine 3E7, chimeric 3E7 or H17, respectively, at 37°C for 1 h. b DB cells were treated with 1 μg/ml RTX and 10 μg/ml murine 3E7, chimeric 3E7 or H17, respectively, at 37°C for 1 h. c Raji-R cells were treated with 10 μg/ml RTX and 10 μg/ml H17 in the presence of 25% NHS for 1, 24 and 48 h. Cell death in a, b, and c was assessed by flow cytometry. d Raji-R cells were treated as in a, then cell death was assessed by Alamar Blue assay and flow cytometry. n the number of independent experiments

To ensure that the killing by H17 in the presence of complement during the 1-h time treatment was true killing, and that cells were not just damaged but able to recover [21], nor artificially reduced in cell count by being cross-linked by H17 [22], Raji-R cells were treated with RTX or RTX+H17 in the presence of NHS for 1, 24 and 48 h at 37°C. Anti-C3b(i) Mab-induced cell aggregates disperse with 24 h of culture [22]. As shown in Fig. 1c, over the 48-h period the enhancement of RTX-mediated killing by H17 was still seen, indicating the enhancing effect observed for H17 on RTX-mediated killing was not transient or artificial. At 48 h, the live cell number in RTX+serum-treated samples was at least six fold higher than that in RTX+H17+serum-treated samples (data not shown) although the percentage of cell death of the former was approaching that of the latter. Also, when Alamar Blue was used to assay cell viability after 1-h CDC, the data showed the same trend as the flow cytometry data (Fig. 1d). There was a slight difference in cell killing between the two methods, since the flow cytometry method monitored membrane integrity while the Alamar Blue assay measured an enzyme activity in live cells.

H17 Preferentially binds to cell-bound C3b(i)

3E7 showed preferential binding to cell-bound C3b(i) as opposed to soluble C3b(i) [22, 31]. To test the binding specificity of H17, Raji-R cells were treated with RTX and H17 in the presence of NHS and various amounts of purified iC3b, and CDC was measured. Under the experimental conditions utilized, about 3.5 μg/ml C3a (MW=8 KD) was generated as measured by a C3a ELISA (Quidel, San Diego, CA, USA), so total C3b+iC3b (both MW=178 KD) would be about 78 μg/ml. Since most C3b molecules are in the C3b(H2O) form [8], total cell-bound C3b+iC3b in the above experiment would be expected to be much lower than 78 μg/ml. In this experiment H17 plus RTX enhanced cell killing by 80% over RTX alone in the absence of competitor (Fig. 2). The addition of soluble iC3b at 90 μg/ml (a higher amount than the total C3b and iC3b generated from RTX+NHS) caused a slight inhibition of the enhancement of cell killing (Fig. 2). The results confirmed that H17, like 3E7, preferentially bound cell surface-associated C3b(i).

Fig. 2.

Fig. 2

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Complement-dependent cytotoxicity of RTX plus H17 is not competed by soluble iC3b. RTX and H17 used in this experiment were both 10 μg/ml; serum: 25%; purified iC3b used here was at 90, 10, 1, 0.1, 0.01 and 0.001 μg/ml. Raji-R cells were treated with RTX, H17 and purified iC3b in the presence of NHS for 1 h at 37°C. Cell death was assayed by flow cytometry

C1q binding and C3b(i) deposition by RTX and H17

We hypothesized that enhancement of CDC by H17 was due to increased complement activation. To determine if the addition of H17 to RTX would induce more complement activation than RTX alone, C1q binding and deposition of C3b(i) molecules on Raji-R treated with RTX and H17 in the presence of NHS were investigated. Flow cytometric analysis demonstrated an average 12-fold increase in C1q binding to cells when H17 was added with RTX in comparison to RTX alone (Fig. 3). The combination of RTX and H17 also induced a three-fold higher level of bound C3b(i) in comparison to RTX alone (Fig. 3). These results support the hypothesis that treatment with H17 in the presence of RTX leads to increased complement activation.

Fig. 3.

Fig. 3

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Addition of H17 increased C1q binding and C3b(i) deposition in RTX-opsonized cells. RTX and H17 at 10 μg/ml were added to Raji-R cells with 25% NHS. Cells were washed and probed for C1q and C3b(i). Shown is the geometric mean of signal intensity on live cells

Killing of non-B lymphocytes by RTX and H17 is minimal

To be useful as an adjunctive therapy, H17-enhanced CDC must be limited to the target cells of RTX, i.e., B cells. To determine if H17 enhancement of RTX-mediated killing is specific for B cells, normal blood lymphocytes were treated with RTX ± H17 in the presence of autologous citrated-plasma (Fig. 4). The same RTX and H17 concentrations as in the cell line experiments were used here. About 90% of normal B cells were killed by RTX during the 1-h treatment, leaving little room for H17 to improve the killing. It was worth mentioning that the killing by serum+H17 in normal B cells was much lower than in NHL cell lines. Malignant cells are known to have membrane features that can trigger IgM-mediated CDC [31] which might explain the observed difference between normal and lymphoma B cells. While normal B cells were sensitive to RTX ± H17 in the presence of complement, CD20 negative T and NK cells were resistant to RTX and RTX plus H17, indicating the killing by RTX ± H17 was specific to B cells.

Fig. 4.

Fig. 4

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Killing of non-B cells by RTX and H17 is minimal. Leukocytes from citrated blood were treated with 10 μg/ml RTX and 10 μg/ml H17 in the presence of autologous citrated-plasma. Cell death of B cells (CD19+) or NK and T cells (CD19−) was assessed by the flow cytometry method

Ex vivo testing of H17 activity on NHL and CLL patient samples

To assess the feasibility of testing H17 efficacy with RTX in a whole animal model, H17 was tested for binding to C3b(i) from various species. C3b(i) from rabbit, rat, guinea pig and dog were not recognized by H17 by ELISA (data not shown). H17 does recognize primate C3b/iC3b, but there is no established primate lymphoma model. Therefore, we have tested H17 efficacy in primary NHL or CLL cells, which are presumed to be closer to the in vivo condition than cell lines.

Mononuclear cells from four bone marrow aspirates were subjected to RTX+H17 treatment in the presence of NHS. In the cases of marginal zone lymphoma (MZL), low-grade NHL and NHL, the addition of H17 increased RTX-mediated tumor cell killing by 50%, 82% and 82%, respectively (Fig. 5a). Enhancement by H17 in one mantle cell lymphoma (MCL) sample could not be measured since treatment with RTX alone resulted in 100% killing (data not shown).

Fig. 5.

Fig. 5

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Enhancement of RTX-mediated killing of NHL and CLL patient samples by H17. a NHL samples; b CLL samples. Samples were treated with RTX and H17 (both at 10 μg/ml) in the presence of NHS. B cells were identified by positive CD19 staining. Cell death was measured by flow cytometry. The numbers at the bottom of the graphs are the numbers of anti-CD20 antibodies per cell expressed in MESF. Most of the samples were assayed in duplicate

Lymph node samples (previously frozen) were of follicular, diffuse large B cell, mantle cell and marginal zone lymphoma types. All the samples were coded and assayed blindly. In seven out of eight samples, after 1-h of treatment at 37°C the majority (over 80%) of the B cells were killed by RTX in the presence of NHS. In all cases, H17 did enhance the killing with RTX, but the increase did not reach statistical significance (data not shown). In samples in which the RTX-mediated cell killing was below 80%, addition of H17 resulted in higher killing (the MCL sample, Fig. 5a).

Enhancement of RTX mediated CDC by H17 in primary CLL cells was also tested. RTX was not as potent at cell killing in the CLL samples as in NHL samples, consistent with clinical observations [15, 18]. However RTX in conjunction with H17 increased the cell killing compared to RTX alone by 9–25% in four out of nine samples. In one of the four samples mentioned above in which H17 showed enhancement (the fifth sample in Fig. 5b), serum+H17 killing observed was the highest among different treatments. We cannot explain the result, though it is possible that somehow a part of the cell pellet was lost during a washing step. Unfortunately, we cannot retest this sample due to the limiting amount of primary cells. In another sample, there was an indication that H17 enhanced RTX-mediated killing, although there were not enough cells to assay it in duplicate (the fourth sample, Fig. 5b). In the sample with the highest CD20 expression, CDC was already 90% by RTX in the presence of NHS, making it difficult to measure enhancement by H17. In another sample which was negative for CD20 expression, neither RTX nor RTX+H17 had any effect. Overall, H17 demonstrated the ability to enhance RTX-mediated killing in about half of the CLL samples (four of nine).

Discussion

Complement-dependent cytotoxicity is a major mechanism of action of RTX in vitro and probably in vivo [4–6, 16, 22, 25]. In two studies using murine models, it was demonstrated that depletion of complement or blocking of complement activation prevented the suppression of tumor growth by RTX treatment [5, 6]. Numerous studies have also demonstrated that RTX promotes robust CDC of B cells in vitro [4, 16, 22, 25].

There is a need for improvement in the efficacy of RTX in treating CD20+ lymphomas [17]. H17 [anti-C3b(i)] is a DeImmunized, chimeric candidate therapeutic Mab capable of enhancing the CDC by RTX. It is shown here that H17 retained high affinity binding activity to C3b(i) and the ability to enhance RTX-mediated killing of NHL B cell lines in the presence of NHS as a source of complement. The cell killing and enhancement observed are complement-dependent, since no killing was observed when cells were treated in the absence of NHS. More importantly, H17 also improved the RTX killing of four out of twelve primary NHL and four out of nine CLL B cell samples ex vivo. The extent of synergy between the two Mabs in vivo is currently unknown, but it could be even greater than that observed ex vivo. Treatment of normal blood lymphocytes with H17 in conjunction with RTX only caused death of B cells, leaving T and NK cells intact. This indicates that the enhancement of RTX-mediated killing by H17 is B-cell specific.

It is reasonable to speculate that H17 enhancement of CDC occurs by direct activation of complement upon binding to C3b(i); H17 may also modulate complement convertase activities (unpublished data, and personal communication with R. P. Taylor and M. J. Glennie). When C1q binding and C3b(i) deposition were measured on cancer cells, it was demonstrated that more complement was indeed activated by H17 plus RTX than RTX alone. For a C3b(i) Mab to be therapeutically useful, binding to target cells must not be strongly competed by soluble C3b(i) molecules. Deposition of C3b(i) on the cell surface via formation of a thio-ester bond should generate a limited number of epitopes different from those expressed on soluble C3b(i) [28, 29] and these neoepitopes could be preferentially recognized by Mab H17. The experiment with purified iC3b (Fig. 2) indeed showed that H17 preferentially recognized cell-bound C3b(i) and killed cells efficiently.

Cancer cells often over-express CRPs [7, 27] hindering the ability of therapeutic tumor-specific Mabs that operate though CDC to lyse these cells [9, 11, 12, 19–21, 32]. A number of different approaches have been tried by other groups to overcome inhibition of complement activation, and thus improve cell lysis by tumor-specific Mabs. These included targeted neutralization of CRPs, use of cocktails of Mabs against multiple epitopes of a tumor-associated antigen, use of secondary antibodies against tumor-specific Mab, and use of soluble β-glycan to trigger iC3b receptor (CR3)-dependent cellular cytotoxicity (see Gelderman et al. [10] and references therein). None of them have yet proved clinically successful.

A unique feature of H17 is that it might be used in conjunction with any complement-activating antibody that promotes deposition of C3b(i), its target molecules, on the tumor cell surface. Therefore, H17 could provide a technology platform to improve the clinical efficacy of any therapeutic antibody that acts through complement activation.

Several reports indicate that antibody-dependent cellular cytotoxicity (ADCC) is involved in RTX-mediated cell lysis in vivo [2, 3, 17, 34] as well as in vitro [16]. The tumoricidal effect of RTX in wild-type mice was much more significant than in FcγRIII deficient mice, which do not have ADCC [3]. Also neutrophil-depleted mice did not response to RTX treatment in a mouse lymphoma model [17]. Moreover, increasing evidence demonstrates that FcγR polymorphisms correlate with positive response to RTX, indicating the potential importance of ADCC as part of the RTX therapeutic mechanism [2, 34]. It is possible that in addition to enhancing CDC, the binding of H17 to human C3b(i) deposited on the cell surface could enhance the interaction between FcγR-bearing cells and cancer cells, hence improving the efficacy of RTX. The extra C3b(i) generated by addition of H17 could also induce complement-dependent cellular cytotoxicity. In those cases, H17 could enhance RTX efficacy in NHL treatment through multiple pathways.

Acknowledgements

We are grateful to Juan Li for her help in developing the Alamar Blue assay. We thank Dr. Ronald P. Taylor for his comments on the manuscript and useful discussion. We also thank Dr. Steven Jones for his comments and help on the manuscript.

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