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. Author manuscript; available in PMC: 2010 Jan 1.
Published in final edited form as: Mol Immunol. 2008 Nov 28;46(3):448–456. doi: 10.1016/j.molimm.2008.10.020

Maleimide conjugation markedly enhances the immunogenicity of both human and murine idiotype-KLH vaccines

Kamran Kafi 1, David J Betting 1, Reiko E Yamada 1, Michael Bacica 3, Kristopher K Steward 1,2, John M Timmerman 1,2
PMCID: PMC2768258  NIHMSID: NIHMS93797  PMID: 19046770

Abstract

The collection of epitopes present within the variable regions of the tumor-specific clonal immunoglobulin expressed by B cell lymphomas (idiotype, Id) can serve as a target for active immunotherapy. Traditionally, tumor-derived Id protein is chemically-conjugated to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde to serve as a therapeutic vaccine. While this approach offered promising results for some patients treated in early clinical trials, glutaraldehyde Id-KLH vaccines have failed to induce immune and clinical responses in many vaccinated subjects. We recently described an alternative conjugation method employing maleimide-sulfhydryl chemistry that significantly increased the therapeutic efficacy of Id-KLH vaccines in three different murine B cell lymphoma models, with protection mediated by either CD8+ T cells or antibodies. We now define in detail the methods and parameters critical for enhancing the in vivo immunogenicity of human as well as murine Id-KLH conjugate vaccines. Optimal conditions for Id sulfhydryl pre-reduction were determined, and maleimide Id-KLH conjugates maintained stability and potency even after prolonged storage. Field flow fractionation analysis of Id-KLH particle size revealed that maleimide conjugates were far more uniform in size than glutaraldehyde conjugates. Under increasingly stringent conditions, maleimide Id-KLH vaccines maintained superior efficacy over glutaraldehyde Id-KLH in treating established, disseminated murine lymphoma. More importantly, human maleimide Id-KLH conjugates were consistently superior to glutaraldehyde Id-KLH conjugates in inducing Id-specific antibody and T cell responses. The described methods should be easily adaptable to the production of clinical grade vaccines for human trials in B cell malignancies.

Keywords: Vaccination, Idiotype, Lymphoma, Tumor Immunity, Antibodies

1. Introduction

The unique collection of epitopes present within the variable regions of tumor-specific immunoglobulin expressed by B cell malignancies (idiotype, or Id) can serve as a target for therapeutic vaccination (Timmerman, 2004). Tumor-specific Id protein can be isolated from individual B cell lymphomas using “rescue hybridization” or molecular cloning techniques, yielding a patient-specific tumor antigen (Levy and Dilley, 1978; Hawkins et al., 1994). Immunization with tumor Id has the capacity to elicit a polyclonal antibody response as well as CD8+ and CD4+ T cells recognizing Id-derived peptides presented at the tumor cell surface on class I and class II MHC proteins, respectively (Timmerman, 2003; Hurvitz and Timmerman, 2005b). While Id proteins are weakly-immunogenic as free Igs, their immunogenicity can be enhanced by chemical linkage to a highly immunogenic foreign carrier protein such as keyhole limpet hemocyanin (KLH) using glutaraldehyde, as shown in numerous studies in both murine models (Maloney et al., 1985; Campbell et al., 1987; Kaminski et al., 1987; Campbell et al., 1988; George et al., 1988; Campbell et al., 1990; Kwak et al., 1996; Timmerman and Levy, 2000a) and human trials (Kwak et al., 1992; Kwak et al., 1995; Hsu et al., 1996; Hsu et al., 1997; Bendandi et al., 1999; Timmerman et al., 2000; Timmerman et al., 2001; Barrios et al., 2002; Timmerman et al., 2002a; Koc et al., 2004; Inoges et al., 2006; Redfern et al., 2006). The efficacy of glutaraldehyde–conjugated Id-KLH protein vaccines in the 38C13 and BCL-1 murine lymphoma models (Campbell et al., 1987; Kaminski et al., 1987; George et al., 1988) led to the translation of this approach to Id vaccine clinical trials in patients with B cell lymphoma (Timmerman and Levy, 2000b). In several phase I/II trials in follicular lymphoma, Id-KLH vaccination has been shown to elicit anti-Id immune responses that correlate with improved progression-free and overall survival (Kwak et al., 1992; Hsu et al., 1997), clearance of circulating tumor cells from the blood (Bendandi et al., 1999), and durable tumor regressions (Timmerman et al., 2002a; Koc et al., 2004; Redfern et al., 2006). Based on these results, three large phase III clinical trials of glutaraldehyde Id-KLH vaccination were initiated in follicular lymphoma patients (Detailed trial information, 2008; Hurvitz and Timmerman, 2005a). These trials all aimed to test whether glutaraldehyde-conjugated Id-KLH co-administered with the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) could prolong progression-free survival after standard cytoreductive chemotherapy or anti-CD20 antibody therapy.

However, recent data suggest that the immunogenicity of traditional glutaraldehyde Id-KLH vaccines is suboptimal. Up to half of patients immunized with glutaraldehyde Id-KLH conjugates do not mount detectable anti-Id immune responses (Hsu et al., 1997; Bendandi et al., 1999; Timmerman et al., 2000; Timmerman et al., 2001; Barrios et al., 2002; Timmerman et al., 2002a; Timmerman et al., 2002b; Koc et al., 2004; Inoges et al., 2006; Redfern et al., 2006). Moreover, in only one of the three recent phase III trials of glutaraldehyde Id-KLH vaccination was a clinical benefit observed over control treatment alone (Biovest Press Release, 2008; Favrille Press Release, 2008; Levy et al., 2008). Glutaraldehyde irreversibly cross-links proteins primarily via lysine residues, with secondary reactions at cysteine, tyrosine, and histidine residues (Migneault et al., 2004). We hypothesized that this extensive cross-linking could damage critical immunogenic epitopes in Id-KLH conjugates and inhibit proteolytic processing of tumor antigen. Furthermore, we proposed that the use of heterobifunctional cross-linking agents containing maleimide groups, which cross-link proteins only via reduced cysteine sulfhydryls, may limit epitope destruction and preserve immunogenicity in Id-KLH vaccines (Yoshitake et al., 1979; Yoshitake et al., 1982; Hashida et al., 1984; Peeters et al., 1989).

We recently described a maleimide-based tumor antigen-carrier protein conjugation technique that markedly enhances the in vivo therapeutic efficacy of Id-KLH vaccines in three different murine B cell lymphoma models (A20, 38C13, and BCL-1)(Betting et al., 2008). This method involves a partial reduction of tumor Id to generate free sulfhydryl groups, followed by reaction with maleimide-activated KLH, yielding conjugates linked by a thioether bond. Maleimide Id-KLH vaccines were found to be superior to glutaraldehyde Id-KLH vaccines in both prophylactic tumor challenge and 4-day early established tumor settings. Anti-tumor effects were mediated by either CD8+ T cells or antibodies, depending on the model employed. While prolonged glutaraldehyde conjugation resulted in progressive loss of immunogenicity, maleimide conjugation yielded potent vaccines regardless of conjugation duration. Unexpectedly, the maleimide (but not glutaraldehyde) Id-KLH linkage was cleavable under lysosomal processing conditions, possibly permitting improved tumor antigen processing and presentation by APCs.

We now report in detail the methods and parameters critical to producing human as well as murine maleimide Id-KLH conjugate vaccines with potent immunogenicity. The level of Id pre-reduction for achieving optimal vaccine efficacy was defined, and maleimide Id-KLH conjugates retained potency even after prolonged frozen storage. Field flow fractionation analysis revealed that maleimide Id-KLH conjugates were far more uniform in size than glutaraldehyde conjugates. Maleimide Id-KLH vaccines were consistently superior to glutaraldehyde Id-KLH vaccines under increasingly stringent therapeutic vaccination conditions against established, disseminated murine lymphoma. Most importantly, human maleimide Id-KLH vaccines were consistently superior to glutaraldehyde Id-KLH vaccines in the induction of humoral and T cell anti-Id responses. These findings are immediately applicable to ongoing attempts at inducing Id-specific immunity against human B cell malignancies.

2. Materials and methods

2.1 Mice and cell lines

BALB/c and C57BL/6 mice (6-10 weeks old) were bred and housed in the Radiation Oncology Barrier Facility, at the University of California, Los Angeles, (UCLA), and experiments were conducted according to UCLA guidelines. The spontaneously arising IgG2a-κ-expressing A20 BALB/c B cell lymphoma line was obtained from the American Type Culture Collection (ATCC)(Rockville, MD)(Kim et al., 1979). Tumor cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal calf serum (FCS)(Omega Scientific, Tarzana, CA), 100 Units/ml penicillin/streptomycin, 2 mM L-glutamine, and 50 mM b-mercaptoethanol (“RPMI complete medium”; all supplements from Invitrogen), at 37°C in 5% CO2.

2.2 Monoclonal immunoglobulins

Murine A20 Id protein (IgG2a-κ) was affinity-purified (protein A) from culture media of a tumor-myeloma cell hybridoma (clone 3D6.3) derived by fusion of A20 lymphoma cells with SP2/0 myeloma cells, as previously described (Betting et al., 2008). The monoclonal antibodies rituximab (human IgG1-κ)(IgG1-R) and trastuzumab (human IgG1-κ)(IgG1-T), were purchased from Genentech (South San Francisco, CA). Monoclonal human IgG3-κ and IgG3-l were purchased from Sigma-Aldrich (St. Louis, MO). Each human monoclonal Ig expresses unique idiotypic determinants, allowing assessment of specific anti-Id immune responses.

2.3 Id-KLH conjugations

Glutaraldehyde conjugations were performed for 15-30 minutes on a rocker platform at room temperature using 1:1 (w:w) mixtures of Id and KLH (Pierce, Rockford, IL) in 0.1% glutaraldehyde (Sigma-Aldrich) as previously described (Campbell et al., 1987). Reactions were carried out until faint, thread-like precipitates began to form, then terminated by dialysis against 1X PBS, pH 7.2 at 4°C.

Maleimide conjugations were performed as diagramed in Figure 1. Monoclonal Igs (1 mg/ml in 1X PBS, pH 7.2) were first reduced in variable concentrations of dithiothreitol (DTT, Sigma-Aldrich) for 1 hour at 37°C, then dialyzed immediately into 1X PBS containing 0.1 M EDTA at 4°C to prevent re-oxidation of sulfhydryl groups. Reduced Id was then mixed with maleimide-activated KLH (Pierce), which was produced using the sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC) heterobifunctional crosslinker (Peeters et al., 1989). Conjugations were carried out at a 1:1 ratio of Id to maleimide-activated KLH (w:w, 1 mg/ml each) for 2 hours at room temperature on a rocker platform, followed by dialysis against 1X PBS at 4°C for 48 hours. The proportion of reduced sulfhydryl groups was quantitated pre-reduction, post-reduction, and post-conjugation using Ellman's reagent (Pierce)(Ellman, 1959). The coupling reaction resulted in complete Id-KLH conjugation, without insoluble precipitate. Completeness of conjugation was confirmed by the absence of detectable free Id protein on non-reducing SDS-PAGE. Conjugates were stored at 4°C and used within 6 weeks unless otherwise indicated. Conjugate stability studies were performed with fresh vaccine conjugates, or vaccines (2 mg/ml total protein, in 1X PBS, pH 7.2) stored at 4°C or -80°C for 6 months prior to use.

Fig. 1.

Fig. 1

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Schematic representation of the maleimide idiotype-KLH carrier protein conjugation process. Tumor-specific Ig (Id) proteins are first treated with DTT to preferentially reduce hinge region disulfides, generating free sulfhydryl groups. Re-oxidation of sulfhydryls is prevented by 0.1 M EDTA. Maleimide-activated KLH is added to reduced Id protein. The maleimide group of the sulfo-SMCC linker reacts with reduced sulfhydryls on the Id to form a stable thioether bond.

2.4 Immunoglobulin reduction studies

For maleimide conjugate reduction level in vivo studies, A20 Id protein (1 mg/ml in 1X PBS, pH 7.2) was pre-reduced in either 0.1 mM, 10 mM, or 300 mM DTT for 1 hour at 37°C, then conjugated as above to maleimide-activated KLH. For Id reduction in vitro studies, aliquots of A20 Id, IgG1-R or IgG1-T (5 mg/ml in 1X PBS, pH 7.2) were treated with a range of DTT concentrations (0.01-300 mM) for 1 hour at 37°C, equilibrated with 1X PBS, pH 7.2 containing 0.1 M EDTA, and reduced sulfhydryl groups per Ig molecule quantitated using Ellman's reagent (Willner et al., 1993).

2.5 Tumor therapy studies

A20 tumor was thawed from common dedicated freezer stocks 3 days before tumor challenge and split the day before use. All mice were injected with tumor on day 0. Mice were given 3 weekly Id-KLH vaccinations starting on day 10 for 5×105 intravenous (i.v.) tumors, and day 14 for 1×106 i.v. tumors. Vaccinations consisted of glutaraldehyde or maleimide Id-KLH conjugates (100 μg total protein: 50 μg Id plus 50 μg KLH, in 100 ml PBS) given subcutaneously (s.c.) in 3 weekly doses in the inguinal crease, alternating sides each week. GM-CSF (55 ng) was co-injected with each Id-KLH vaccine, and at the identical site daily for 3 consecutive days as an adjuvant (Kwak et al., 1996) Control treatments consisted of 100 μl of Hank's balanced salt solution (HBSS)(Invitrogen). Mice were followed for survival, and sacrificed when appearing moribund, per institutional guidelines.

2.6 Long-term stability of Id-KLH conjugates

A20 maleimide Id-KLH conjugates (1 mg/ml Id) were stored for 6 months at 4°C or -80°C in 1X PBS, pH 7.2, and the integrity of the maleimide linkage was compared to freshly conjugated maleimide Id-KLH by SDS-PAGE analysis under non-reducing conditions on a 12% Precise Protein Gel (Pierce).

2.7 Id-KLH conjugate particle size analysis

To determine the distribution of Id-KLH particle sizes, field flow fractionation analysis was performed using an Eclipse AFFF system (Wyatt Technology, Santa Barbara, CA). This device splits the flow of an isocratic Agilent HPLC pump to create focusing conditions for particle separation based on differences in diffusion coefficients as particles repel an accumulation wall. The accumulation wall consisted of a low protein-binding regenerated cellulose membrane with 10 kDa molecular weight cutoff over a porous metal surface. Samples (20 ml) were injected at a cross-flow rate 1.5 ml/min to detect Ig fragments, intact monomers and smaller aggregates, and cross-flow rate then decreased to 0.1 ml/min over 16 minutes for larger aggregates and particulates. Samples were eluted in 1X PBS, pH 7.2 with 0.02% sodium azide, and elution monitored at 280 nm by an Agilent UV diode-array detector, Optilab refractive index detector, and DAWN EOS light scattering detector (Wyatt Technology, Santa Barbara, CA).

2.8 Vaccination with human Id-KLH conjugates

Glutaraldehyde and maleimide conjugates were prepared with 4 monoclonal human IgGs (IgG1-R, IgG1-T, IgG3-κ and IgG3-λ). Groups of 4 mice were vaccinated s.c. with the Id-KLH conjugates plus GM-CSF as above on days 0, 14, and 28. Ten days later, mice were bled for immune sera for measurement of humoral immune responses.

2.9 Quantitation of anti-idiotype antibodies by ELISA

F(ab′)2 fragments were prepared for each of the human IgGs with the F(ab′)2 preparation kit (Pierce), and coated onto 96-well Nunc-immuno Maxisorp ELISA plates (5 μg/ml)(Nunc, Rochester, NY). F(ab′)2 fragments were used as ELISA targets to minimize reactivity of immune sera to human Fc constant region determinants, thus focusing the detected response on variable region (idiotypic) epitopes. Pooled sera from mice vaccinated with each IgG were added to plates coated with each purified F(ab′)2 protein and serially diluted. A polyclonal mouse anti-human IgG (H+L specific, Pierce) antibody was used as a standard. Anti-Id antibodies were detected using horseradish peroxidase (HRP)-conjugated anti-mouse IgG (γ-specific, Southern Biotech, Birmingham, AL). Absorbance was determined with the 2,2′-azinobis(3-ethyl)-benzthiazoline sulfonic acid (ABTS, Sigma-Aldrich) substrate at 405 nm using a SPECTRAmax Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA). Data was analyzed using SoftMax® Pro Version 5.0 software (Molecular Devices).

2.10 Antigen-specific T cell proliferation assays

Groups of 4 mice were vaccinated s.c. with maleimide or glutaraldehyde IgG1-T-KLH conjugates plus GM-CSF as above on days 0, 14, and 28. Ten days later, splenocytes were harvested and seeded in quadruplicate into 96-well U-bottom plates (Nunc) at 2×105 cells/well in RPMI complete medium. Graded concentrations of KLH (1, 10, 100 μg/ml), F(ab′)2 IgG1-T (1 or 10 μg/ml), or control F(ab′)2 IgG1-R (1 or 10 μg/ml) proteins were added to the wells, and incubated at 37°C in 5% CO2 for 4 days. Cells were pulsed with 1 μCi/well 3[H]-thymidine (MP Biomedicals, Solon, OH), and harvested 16 hours later. Incorporated radioactivity (counts per minute, cpm) was measured using a β-liquid scintillation analyzer (PerkinElmer, Waltham, MA), and results from quadruplicate cultures reported as arithmetic means converted to a stimulation index (experimental cpm divided by media alone cpm).

2.11 Statistical Analysis

Survival differences among groups of mice were assessed using the Kaplan-Meier method with the log-rank test using Prism software (Graph-Pad Software, San Diego, CA). P values were considered statistically significant at p less than 0.05.

3. Results

3.1. Optimal maleimide Id-KLH conjugate vaccine efficacy is achieved using pre-reduction of hinge region disulfides with 0.1 mM DTT

A key step in the production of maleimide-based tumor antigen-carrier protein conjugates is a partial pre-reduction of cysteine residues in the tumor antigen to generate free sulfhydryl groups that can then react with maleimide-activated KLH (Figure 1). We previously found that maleimide conjugate vaccines produced using pre-reduction with 0.1 mM DTT induced potent tumor protection in three different murine lymphoma models (Betting et al., 2008). These reduction conditions were chosen as they preferentially reduce the more exposed hinge region disulfide bonds (Gunewardena and Cooke, 1966; Willner et al., 1993). To estimate the number of free sulfhydryl groups generated under various reducing conditions, we subjected A20 Id and several human monoclonal Igs to a range of DTT concentrations (0.01 – 300 mM), and determined their free sulfhydryl content using Ellman's reagent (Figure 2A). All three Ids showed similar behavior in these conditions, with 0.1 mM DTT yielding approximately four sulfhydryl groups per Ig molecule, likely corresponding to reduction of the two hinge region disulfides (Gunewardena and Cooke, 1966; Willner et al., 1993). Reduction with lower than 0.1 mM concentrations of DTT led to incomplete Id conjugation to maleimide-activated KLH and was therefore not explored in animal experiments (data not shown). To determine the optimal level of antigen pre-reduction, we produced A20 maleimide Id-KLH conjugates after exposure to different levels of DTT (0.1, 10, and 300 mM). As shown in Figure 2B, Id-KLH produced using 0.1 mM DTT offered the highest level of tumor eradication (50% vs. 12.5% for 10 and 300 mM).

Fig. 2.

Fig. 2

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Effects of reduction level on free sulfhydryl content of Igs and vaccine efficacy. (a) Susceptibility of human and murine monoclonal Ig preparations to DTT reduction. Human IgG1-R, IgG1-T, and murine IgG2a (A20 Id) were exposed to varying concentrations of DTT (0.01, 0.1, 1, 5, 10, 50, 100, 300 mM) for 1 hour at 37°C, followed by buffer exchange into 1X PBS plus 0.1M EDTA, and free sulfhydryl (SH) content was determined via Ellman's test. (b) Effect of Id reduction conditions before maleimide conjugation on Id-KLH vaccine efficacy in vivo. A20 Id was reduced using 0.1, 10, or 300 mM DTT, then conjugated to maleimide-activated KLH. Groups of mice (n = 8) were inoculated s.c. with 1×105 A20 tumor cells on day 0, and on day 4 began 3 weekly immunizations with the respective conjugates plus GM-CSF, or control treatment with HBSS, and followed for survival. Data are representative of two independent experiments.

3.2 Potency and stability of maleimide Id-KLH conjugates is maintained after long-term frozen storage

The large-scale production of maleimide Id-KLH vaccines for human trials would necessitate long-term storage of conjugates during periods of transport and vaccination courses. To test the long-term stability of maleimide Id-KLH conjugates, we compared A20 maleimide Id-KLH conjugates stored at either 4°C or frozen for 6 months to a freshly conjugated vaccine. After 6 months, non-reducing SDS-PAGE analysis of the sample stored at 4°C demonstrated that a fraction (approximately 25%) of the Id was no longer bound to KLH, while Id and KLH remained completely bound in the freshly-prepared and frozen conjugates (Figure 3A). The in vivo efficacy of these conjugates was then evaluated in tumor bearing mice (Figure 3B). Both fresh and frozen conjugates eradicated tumor from 50% of mice, with significantly improved survival over treatment with HBSS (p < 0.0001), while the conjugate stored at 4°C did not induce significant protection (12.5%, p = 0.057). This loss of immunologic potency in the sample stored at 4°C correlated with partial release of Id from the conjugate (Figure 3A). Thus, to retain optimal potency during long-term storage, maleimide Id-KLH vaccines should be stored frozen.

Fig. 3.

Fig. 3

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Stability and immunologic potency of maleimide Id-KLH conjugates is maintained after long-term frozen storage. A20 conjugates (1mg/ml Id in 1X PBS, pH 7.2) were stored for 6 months at 4°C or -80°C, and compared with freshly conjugated Id-KLH. (a) Long-term stability of frozen maleimide Id-KLH. Conjugates (10 μg) were subjected to non-reducing SDS-PAGE alongside 5 μg of free A20 Id. The absence of free Id in the freshly-conjugated or -80°C stored lane indicates that Id is completely conjugated to the carrier protein and thus stable under these storage conditions. Some free Id is seen in the sample stored at 4°C for 6 months, indicating that the Id-KLH linkage is less stable under these conditions. (b) In vivo efficacy of maleimide Id-KLH conjugates after long term storage. Mice (n = 8) were inoculated s.c with 1×105 A20 cells on day 0, and on day 4 began 3 weekly immunizations with the respective conjugates plus GM-CSF, or HBSS, and followed for survival.

3.3 Maleimide conjugation yields human and murine Id-KLH conjugates of more uniform size than glutaraldehyde conjugates

Prolonged conjugation of murine or human Id proteins to KLH with glutaraldehyde can result in large, insoluble aggregates with possible loss of important immunogenic epitopes (Betting et al., 2008). In contrast, with maleimide conjugation we did not observe the formation of the large, thread-like, insoluble precipitates seen with glutaraldehyde. All but one of our five maleimide conjugates remained clear after the conjugation reaction; only with human IgG1-R did we observe a moderate degree of turbidity after conjugation. To objectively characterize the physical differences between Id-KLH conjugates, we utilized the technique of field flow fractionation. This flow-based biophysical method has been used to analyze protein aggregates in therapeutic monoclonal antibody preparations (Liu et al., 2006). As illustrated in Figure 4, maleimide conjugates were relatively uniform in their particle size distributions, though IgG1-R had the largest particles, accounting for its observed turbidity. In contrast, glutaraldehyde conjugates displayed a marked heterogeneity in particle size distributions, with a dominance of larger aggregates in the case of IgG1-R, in which insoluble precipitates were readily visible.

Fig. 4.

Fig. 4

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Field flow fractionation (FFF) analysis of Id-KLH particle sizes after maleimide versus glutaraldehyde conjugation. Maleimide (upper panel) and glutaraldehyde (lower panel) Id-KLH conjugates of human IgG1-R, IgG1-T, and murine IgG2a-A20 Id were subjected to FFF to determine the proportion (cummulative weight fraction) of conjugate protein particles of the indicated sizes (root mean square [rms] radius in nm).

3.4. Maleimide Id-KLH conjugate vaccine maintains efficacy over glutaraldehyde Id-KLH under increasingly stringent conditions of established lymphoma

In our earlier study, we reported that maleimide Id-KLH conjugates elicit superior anti-tumor immunity compared to glutaraldehyde conjugates against 3 different murine B cell lymphomas in prophylactic tumor challenge and 4-day early established s.c. tumor settings (Betting et al., 2008). To extend our efficacy comparison of the conjugates, we utilized the A20 model, where maleimide versus glutaraldehyde Id-KLH vaccines were tested under increasingly stringent conditions (Figure 5). In mice bearing 10-day established i.v. tumors, maleimide Id-KLH again eradicated tumor from a greater proportion of mice than did glutaraldehyde (50% vs. 12.5%)(Figure 5A). It has been previously shown that i.v. injection with as few as 106 A20 lymphoma cells induces tumor-specific T cell tolerance after a period of 10 days (Sotomayor et al., 1999). We treated mice with maleimide or glutaraldehyde Id-KLH vaccines 14 days after i.v. injection of 106 A20 lymphoma cells (Figure 5B). In this setting, glutaraldehyde Id-KLH did not affect survival, while maleimide Id-KLH extended survival in all animals, and eradicated tumor from 37.5% (p = 0.0004). Thus, even under the most clinically-relevant conditions of advanced, disseminated tumor, maleimide Id-KLH remained superior to a traditional glutaraldehyde Id-KLH vaccine.

Fig. 5.

Fig. 5

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The efficacy advantage of maleimide over glutaraldehyde Id-KLH vaccines is maintained over increasingly stringent conditions of established A20 lymphoma. (a) Eradication of day 10-established, disseminated A20 lymphoma. Groups of mice (n = 8) were inoculated i.v. with 5×105 A20 tumor cells on day 0, and on day 10 began 3 weekly s.c. immunizations with maleimide or glutaraldehyde Id-KLH conjugates plus GM-CSF, or HBSS control. Mice were sacrificed when moribund. (b) Eradication of day 14-established, disseminated A20 lymphoma. Mice were treated as above, except for i.v. inoculation with 1×106 A20 tumor cells, and vaccinations beginning on day 14.

3.5 Human maleimide Id-KLH vaccines induce superior anti-Id antibody and T cell responses compared to glutaraldehyde conjugates

As a prelude to the clinical testing of maleimide Id-KLH vaccination in humans, we wished to assess the immunogenicity of human Id-KLH conjugates in vivo. Four different human monoclonal Igs of the isotypes used in recombinant Id vaccine trials (IgG1 and IgG3)(Timmerman, 2004) were conjugated to KLH using maleimide or glutaraldehyde, and used to vaccinate groups of mice. Anti-idiotypic antibodies in the sera of vaccinated mice were measured by ELISA, using F(ab′)2 fragments of each immunogen as targets to minimize detection of serum antibodies against shared xenogeneic determinants in the human IgG constant regions (Figure 6). In all four cases, maleimide Id-KLH vaccines elicited higher (2.1 to 14.2-fold) anti-Id antibody titers than the corresponding glutaraldehyde Id-KLH conjugates. Interestingly, antibodies elicited by glutaraldehyde conjugates reacted to a greater degree with control Igs (Figure 6A, B, D) than those elicited by the corresponding maleimide conjugates. In contrast, nearly all of the reactivity in the sera of mice vaccinated with maleimide conjugates was directed at idiotypic determinants, as there was little cross-reaction with the three other control human Igs.

Fig. 6.

Fig. 6

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Human Maleimide Id-KLH vaccines induce higher anti-Id antibody titers than glutaraldehyde conjugates. Groups of mice (n = 4) were given 3 bi-weekly s.c. vaccinations with either maleimide or glutaraldehyde Id-KLH and bled 10 days after the final vaccination. Sera from each group were pooled and anti-Id levels measured by ELISA using plates coated with F(ab′)2 fragments of (a) IgG3-κ, (b) IgG3-λ, (c) IgG1-T, or (d) IgG1-R proteins. Error bars represent the mean and standard deviation of 3 replicate determinations. These data are representative of two individual experiments.

To evaluate the induction of T cell responses, mice were vaccinated with maleimide or glutaraldehyde conjugates of IgG1-T-KLH, and splenocytes tested for proliferation against KLH, IgG1-T, or an isotype control Ig (Figure 7). Splenocytes from mice immunized with both the maleimide and glutaraldehyde conjugates proliferated against KLH (left panel), though the response in the maleimide group was substantially stronger. Idiotype-specific proliferation was only seen in the splenocytes from mice immunized with the IgG1-T maleimide conjugate; these splenocytes displayed a higher degree of proliferation to the specific immunogen (IgG1-T, center panel) than to an isotype control Ig antigen (center panel). These results demonstrate the induction of superior T cell proliferative responses by maleimide versus glutaraldehyde human Id-KLH conjugates.

Fig. 7.

Fig. 7

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Maleimide conjugation of human Id improves the T cell response to both Id and KLH. Groups of mice (n = 4) were given 3 bi-weekly s.c. vaccinations with IgG1-T conjugated to KLH with either maleimide or glutaraldehyde, and splenocytes collected and pooled 10 days after the final vaccination. Splenocytes were cultured for 4 days with either KLH, F(ab′)2 fragments of IgG1-T, or an isotype control Ig (IgG1-R) at the indicated concentrations (μg/ml). Proliferation was measured by 3[H]-thymidine incorporation after a 16 hour pulse, and data represented as stimulation index = experimental counts per minute (cpm) divided by media alone cpm.

4. Discussion

Targeting the Id of B cell malignancies remains an attractive strategy for the active immunotherapy of cancer. This tumor-specific antigen can be obtained with relative ease from B cell lymphomas using hybridoma or recombinant DNA technologies (Timmerman, 2004). The challenge however, is to deliver the Id to the immune system with minimal epitope destruction in order to elicit maximal humoral and T cell responses. Conjugation of the weakly-immunogenic self-derived Id to a highly immunogenic carrier protein such as KLH is an established means of improving immune recognition (Kaminski et al., 1987; Kwak et al., 1992; Timmerman and Levy, 2000a). In over 20 years of investigational use, glutaraldehyde has served as the standard for Id-KLH conjugation in both laboratory and clinical studies (Kaminski et al., 1987; Timmerman and Levy, 2000b). While there is compelling evidence for clinical benefit in some studies of therapeutic Id vaccination against follicular lymphomas (Kwak et al., 1992; Hsu et al., 1996; Bendandi et al., 1999; Timmerman et al., 2002a), many patients lack apparent immune responses or clinical benefits using traditional glutaraldehyde Id-KLH vaccines (Hsu et al., 1997; Timmerman et al., 2000; Timmerman et al., 2001; Barrios et al., 2002; Timmerman et al., 2002a; Timmerman et al., 2002b; Koc et al., 2004; Inoges et al., 2006; Redfern et al., 2006; Levy et al., 2008). We recently described the superiority of maleimide over glutaraldehyde Id-KLH conjugates as therapeutic vaccines against three different murine lymphoma models (Betting et al., 2008). Notably, glutaraldehyde Id-KLH conjugation could result in “over-conjugation” of the tumor antigen, yielding conjugates with decreased efficacy, while the heterobifunctional maleimide cross-linker chemistry yielded potent vaccine product irrespective of conjugation duration. Given these results, we suggest that in recent trials of glutaraldehyde Id-KLH vaccines, the potential effectiveness of these vaccines may have been compromised by suboptimal protein cross-linking.

In preparation for clinical translation, we have now explored in further detail the conditions and parameters important for achieving superior in vivo immunogenicity with human as well as murine maleimide Id-KLH vaccines. An important step in the maleimide tumor antigen-carrier protein conjugation process is the reduction of cysteine residues to generate free sulfhydryl groups. A one-hour incubation of Id with 0.1 mM DTT is known to preferentially reduce hinge regions disulfides (Gunewardena and Cooke, 1966), thus leaving the variable region (Id) domains of the Ig intact. In the current study, human IgGs behaved similarly to the murine A20 lymphoma Id when treated with varying concentrations of DTT. The 0.1 mM DTT concentration yielded approximately 4 reduced sulfhydryl groups per Ig molecule (Figure 2A). Since lower DTT concentrations resulted in fewer free sulfhydryls and incomplete carrier protein conjugation, we thus considered 0.1 mM DTT as optimal for maleimide Id-KLH conjugation. Moreover, the Id-KLH vaccines produced using 0.1 mM DTT offered optimal anti-tumor immunity in vivo (Figure 2B).

When producing therapeutic Id-carrier protein conjugate vaccines for clinical use, manufacturing methodologies yielding vaccines with consistent immunologic potency and purity are desirable. Here we demonstrated that frozen conjugates remained completely intact after 6 months of storage, while the 4°C sample showed a partial release of Id from KLH (Figure 3A). This result correlated with the maleimide Id-KLH conjugate's ability to eradicate lymphoma in vivo (Figure 3B). Although we have found that A20 Id-KLH vaccines are effective when used up to 6 weeks after preparation and storage at 4°C, vaccine potency is significantly diminished after 6 month storage at 4°C. Therefore, the maleimide Id-KLH linkage can be labile under certain conditions, and during long-term storage, maleimide Id-KLH vaccines should be stored frozen.

In addition to improving efficacy, maleimide conjugation also yields vaccines with more uniform composition than glutaraldehyde. The homofunctional cross-linking agent glutaraldehyde can lead to undesired Id-Id and KLH-KLH conjugates devoid of anti-tumor activity (Kaminski et al., 1987; Timmerman and Levy, 2000b). Furthermore, the glutaraldehyde reaction proceeds progressively, eventually leading to large aggregates of cross-linked protein, possibly with diminished immunogenicity (Betting et al., 2008). In the original Id vaccine clinical trials at Stanford, Id-KLH vaccines were prepared individually with the duration of glutaraldehyde conjugation titrated until the initial formation of visible precipitates in attempt to achieve an optimal level of conjugation (Kwak et al., 1992; Timmerman et al., 2002a). However, recent phase III trials of glutaraldehyde Id-KLH vaccines have utilized a fixed 1-2 hour conjugation period, at times resulting in variable amounts of insoluble precipitated material (J. Timmerman, unpublished observations). We sought an objective biophysical method for characterizing the particle sizes of Id-KLH conjugates. Using the technique of field flow fractionation, we found that glutaraldehyde Id-KLH conjugates varied widely in their particle size distributions, while those of maleimide conjugates were far more uniform (Figure 4). Thus, in addition to enhanced efficacy, the uniformity of product obtained with maleimide Id-KLH conjugation should offer advantages in the large-scale manufacturing and quality control of Id-KLH vaccines. Futhermore, the particulate nature of Id-KLH conjugates may itself enhance immunogenicity, as particulate antigens often induce greater T cell immunity than smaller, monomeric forms of protein antigens (Falo et al., 1995; Xiang et al., 2006). Maleimide conjugation consistently yielded particle sizes believed to be associated with favorable induction of T cell responses (40-150 nm)(Xiang et al., 2006).

In addition to performing a more detailed molecular characterization of maleimide Id-KLH vaccines, we have now confirmed their superiority over glutaraldehyde conjugates in several important in vivo settings. In a stringent test against 14-day established, disseminated lymphoma, glutaraldehyde Id-KLH co-administered with adjuvant GM-CSF offered no in vivo vaccine efficacy (Figure 5). However, maleimide Id-KLH plus GM-CSF was able to improve survival and eradicate some tumors in this clinically-relevant setting. Thus, the maleimide Id-KLH, but not the glutaraldehyde vaccine was apparently able to overcome tumor-specific T cell tolerance (Sotomayor et al., 1999). More importantly, in this study we have confirmed that the in vivo immunogenicity of a panel of human Id proteins conjugated to KLH via maleimide is superior to that of glutaraldehyde conjugates (Figure 6). Intriguingly, while some human glutaraldehyde conjugates could elicit substantial levels of anti-Id antibodies, the responses were heterogenous, and the levels consistently lower than those obtained with maleimide conjugates. Such heterogeneity may reflect the variation observed in immune and clinical responses among lymphoma patients treated with glutaraldehyde Id-KLH vaccines (Hsu et al., 1997; Timmerman et al., 2002a; Weng et al., 2004). As such, maleimide conjugation, with its uniformly higher immune response rate, could be expected to add to the clinical potency of human Id-KLH vaccines. In this xenogeneic immunization setting it was not possible to assess the induction of CTL activity against human Ids. However, in our previous studies in the A20 murine lymphoma model, heightened CD8+ T cell activity against A20 Id was associated with increased levels of anti-Id antibodies after maleimide Id-KLH vaccination (Betting et al., 2008). Thus, it is reasonable to suggest that maleimide Id-KLH may provide more effective CTL responses against human tumor Id proteins as well. Moreover, the T cell proliferative response of murine splenocytes after vaccination with a human maleimide Id-KLH conjugate was superior to that after glutaraldehyde conjugate immunization, reinforcing once more the advantage of maleimide conjugation for eliciting Id-specific T cell responses (Figure 7).

The success of a therapeutic tumor antigen vaccine is certainly expected to depend on the summation of a number of factors not only involving the tumor antigen composition but also the immunologic interplay between the host and the tumor itself. In the case of Id-carrier protein vaccines for B cell malignancies, our data suggest that sulfhydryl-based maleimide conjugation offers a substantial improvement over traditional glutaraldehyde conjugation. Through exploitation of this chemistry, relevant antigenic structures are better preserved (Betting et al., 2008). Our repeated finding of the superiority of maleimide over glutaraldehyde Id-KLH vaccines against both human and murine Id proteins provides strong rationale for the clinical testing of this approach against human B cell cancers. Maleimide-based linkers should be easy to substitute for glutaraldehyde, having already been used to produce immunotoxins for human trials (Vitteta and Thorpe, 1991; Grossbard et al., 1992). In the current study, commercially-available maleimide-activated KLH was utilized for convenience. We have since produced maleimide-activated KLH in our laboratory using the sulfo-SMCC linker (data not shown). Incubation of free KLH with 10-fold molar excess sulfo-SMCC (assuming a MW for KLH of 5×106 Da, whereby 1 μM KLH is equal to 5 mg/ml) for one hour yielded maleimide-activated KLH that conjugated completely to a variety of human Ig preparations (Betting et al., 2008). Thus, maleimide Id-KLH conjugation (or conjugation of tumor antigens to alternative immunogenic carrier proteins), as we have defined in the current report, should be easily adaptable to the production of clinical grade vaccines for human trials. We believe that our molecular and immunologic characterization of maleimide Id-KLH vaccines represents an important step towards the goal of inducing Id-specific tumor immunity in a high frequency of patients with B cell malignancies.

Acknowledgments

We thank Dr. Sherie Morrison (UCLA) for critical evaluation of the manuscript.

Supported by the Leukemia & Lymphoma Society Translational Research Program grant #6192-06. JMT is a Damon Runyon Clinical Investigator supported in part by the Damon Runyon Cancer Research Foundation (CI-26-05). KKS was supported by the National Institutes of Health Institutional Training Grant T32-CA009120-31.

Footnotes

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