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The Journal of Immunology, 2008, 181, 4131 -4140
Copyright © 2008 by The American Association of Immunologists, Inc.
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Sulfhydryl-Based Tumor Antigen-Carrier Protein Conjugates Stimulate Superior Antitumor Immunity against B Cell Lymphomas1

David J. Betting, Kamran Kafi, Alireza Abdollahi-Fard, Sara A. Hurvitz and John M. Timmerman2

Division of Hematology and Oncology, Department of Medicine, University of California, Los Angeles, Los Angeles, CA 90095


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Therapeutic vaccination of B cell lymphoma patients with tumor-specific Ig (idiotype, or Id) chemically coupled to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde has shown promising results in early clinical trials, and phase III trials are underway. However, glutaraldehyde Id-KLH vaccines fail to elicit anti-Id immune and clinical responses in many patients, possibly because glutaraldehyde reacts with lysine, cysteine, tyrosine, and histidine residues, damaging critical immunogenic epitopes. A sulfhydryl-based tumor Ag-carrier protein conjugation system using maleimide chemistry was used to enhance the efficacy of Id-KLH vaccines. Maleimide Id-KLH conjugates eradicated A20 lymphoma from most tumor-bearing mice, whereas glutaraldehyde Id-KLH had little efficacy. Maleimide Id-KLH elicited tumor-specific IgG Abs and T cells, with CD8+ T cells being the major effectors of antilymphoma immunity. Maleimide Id-KLH vaccines also demonstrated superior efficacy in 38C13 and BCL-1 lymphoma models, where Abs were shown to be critical for protection. Importantly, standard glutaraldehyde Id-KLH conjugation procedures could result in "overconjugation" of the tumor Ag, leading to decreased efficacy, whereas the heterobifunctional maleimide-based conjugation yielded potent vaccine product regardless of conjugation duration. Under lysosomal processing conditions, the Id-carrier protein linkage was cleavable only after maleimide conjugation. Maleimide KLH conjugation was easily performed with human Igs analogous to those used in Id-KLH clinical trials. These data support the evaluation of sulfhydryl-based Id-KLH vaccines in lymphoma clinical trials and possibly the use of tumor Ag-carrier protein vaccines for other cancers.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Therapeutic vaccines targeting the tumor-specific Ig (idotype, or Id) clonally expressed by B cell lymphomas have reached an advanced state of clinical development (1). Tumor-specific Id protein can be derived from individual B cell lymphomas using hybridoma (2) or molecular cloning (3) techniques, yielding a custom-made tumor Ag for each patient. Vaccination with tumor-derived Id can potentially elicit a polyclonal Ab response, as well as CD8+ and CD4+ T cells recognizing Id-derived peptides presented on class I and class II MHC proteins at the tumor cell surface (1, 4). To render this self-derived protein more recognizable to the immune system, Id is usually chemically conjugated to the highly immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH)3 using glutaraldehyde, as performed in numerous studies in both mouse models (5, 6, 7, 8, 9, 10, 11, 12) and humans (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). The effectiveness of glutaraldehyde-conjugated Id-KLH protein vaccination in several murine lymphoma models (6, 7, 8) led to its adoption in Id vaccine clinical trials in patients with B cell lymphoma (25). In phase I/II trials in lymphoma, Id-KLH vaccination has been shown to elicit anti-Id immune responses that correlate with improved progression-free and overall survival (13, 16), clearance of circulating tumor cells from the blood (17), and durable tumor regressions (20, 22, 24). Based on these results, three large phase III clinical trials of Id-KLH vaccination are now underway in North America (26). All three trials are testing the ability of glutaraldehyde-conjugated Id-KLH coadministered with the cytokine GM-CSF to delay tumor regrowth after standard cytoreductive therapy (26) (detailed trial information is available from Genitope (www.genitope.com), Favrille (www.favrille.com), and Accentia (www.accentia.net)).

However, glutaraldehyde Id-KLH conjugates fail to elicit detectable anti-Id immune responses in up to one-half of immunized patients (16, 17, 18, 19, 20, 21, 22, 23, 24, 27). Although tumor-induced tolerance and immunosuppression likely contribute to this suboptimal response rate, poor vaccine immunogenicity may also be a critical factor. For example, in some murine B cell lymphoma models such as A20, glutaraldehyde Id-KLH vaccines have been reported to lack antitumor efficacy (28, 29). Glutaraldehyde primarily cross-links proteins via lysine and cysteine residues, with secondary reactions at tyrosine and histidine (30). This extensive cross-linking could potentially destroy critical immunogenic epitopes and inhibit proteolytic processing of the tumor Ag. The amino acid sequence of A20 Id contains numerous glutaraldehyde-reactive residues within and adjacent to two predicted class I MHC-binding nonapeptides that may serve as targets for tumor Ag-specific CD8+ T cells (31). Thus, cross-linking via glutaraldehyde could hinder processing of or directly damage epitopes in this and other Id proteins. In contrast, heterobifunctional cross-linking agents containing maleimide groups cross-link proteins only via reduced cysteine sulfhydryl groups, thus limiting the potential for epitope destruction (32, 33, 34, 35). We hypothesized that the epitopes of some Id proteins, including that of the A20 lymphoma, might be better preserved using the sulfhydryl-based maleimide conjugation method, yielding a vaccine with more potent antitumor immunity.

To test this hypothesis, maleimide Id-KLH conjugation was compared with glutaraldehyde conjugation in the A20 murine B cell lymphoma model. Maleimide conjugates were strikingly potent in their ability to eradicate A20 lymphoma from tumor-bearing mice, whereas glutaraldehyde Id-KLH had little efficacy. Maleimide Id-KLH vaccines also showed superior efficacy over glutaraldehyde Id-KLH in the 38C13 and BCL-1 lymphoma models, upon which most current clinical trials are based (25). Antitumor effects were mediated by CD8+ T cells or Abs depending on the model used. Importantly, standard glutaraldehyde Id-KLH conjugation procedures could result in "overconjugation" of the tumor Ag, with attendant loss of immunogenicity. The Id-KLH linker was cleavable under lysosomal conditions only after maleimide, but not after glutaraldehyde conjugation. These data support the testing of sulfhydryl-based Id-KLH vaccines in clinical trials for B cell malignancies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and cell lines

BALB/c and C3Hf/Sed/Kam mice (6–10 wk old) were bred and housed in the Radiation Oncology Barrier Facility at the University of California, Los Angeles, CA (UCLA), and experiments were conducted according to UCLA guidelines. The spontaneously arising IgG2a/{kappa}-expressing A20 BALB/c B cell lymphoma line was obtained from the American Type Culture Collection (36). 38C13 (a gift from R. Levy, Stanford University, Stanford, CA) is a C3H B cell lymphoma expressing surface IgM/{kappa} (37). BCL-1 (a gift from S. Strober, Stanford University) is a spontaneously arising BALB/c B cell lymphoma expressing surface IgM/{lambda} (38). Tumor cell lines were found to be free of mycoplasma using the Safe Cells kit (Bionique Testing Laboratories). Tumor cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated FCS (Omega Scientific), 100 U/ml penicillin/streptomycin, 2 mM L-glutamine, and 50 µM 2-ME (all from Invitrogen) at 37°C in 5% CO2.

Antibodies

38C13 (38C13-A1.2, IgM/{kappa}) and BCL-1 (6A5.1, IgM/{lambda}) Id proteins were purified from hybridomas as previously described (6, 39). Anti-Id Abs S1C5 (38C13 anti-Id, mouse IgG2a) and 1G6 (A20 anti-Id, IgG1) (a gift from R. Levy) were purified from culture supernatants (5, 40). T cell-depleting Abs HB129 (anti-CD8{alpha}, mouse IgG2a), GK1.5 (anti-CD4, rat IgG2b), and 53.6.72 (anti-CD8{alpha}, rat IgG1) were purchased from BioExpress. Control rat IgG and mouse IgG2a (UPC-10) were purchased from Sigma-Aldrich.

Generation of A20 Id-producing hybridoma

The A20 Id-producing hybridoma was generated in our laboratory as previously described (41). Briefly, BrdU-resistant (aminopterin-sensitive) A20 cells were fused with SP2/0 myeloma cells, hypoxanthine/aminopterin/thymidine (HAT)-resistant cells secreting IgG2a were cloned by limiting dilution, and high-producing clone 3D6.3 was selected. The cDNAs encoding the Ig variable regions of 3D6.3 were amplified, sequenced, and found to have complete identity to parental wild-type A20. 3D6.3 was grown in PFHM II protein-free hybridoma medium (Invitrogen) and Id was purified from culture supernatants using protein A Fast Flow columns (Amersham Biosciences).

Id-KLH conjugations

Fig. 1 illustrates the conjugation chemistries for the two cross-linking agents used in this study. Glutaraldehyde conjugations were generally performed for 15–30 min on a rocker at room temperature using 1:1 (w/w) mixtures of Id:KLH in 0.1% glutaraldehyde (Sigma-Aldrich) as previously described (7). Conjugations were conducted until a faint precipitate began to form, then terminated by dialysis against 1x PBS at 4°C. For glutaraldehyde time course studies, Id proteins were conjugated to KLH for 0, 2, 15, 30, 60, or 120 min before dialysis. For maleimide conjugations, Id proteins (1 mg/ml in PBS) were reduced in 0.1 mM DTT (Sigma-Aldrich) for 1 h at 37°C and dialyzed into 1x PBS containing 0.1 M EDTA to prevent reoxidation of sulfhydryl groups. Under these conditions, reduction was partial (~50%). Maximal (100%) reduction levels were established using 300 mM DTT and boiling for 10 min. Reduced Id was conjugated to maleimide-activated KLH (Pierce) at a 1:1 ratio of Id to KLH (w/w) (1 mg/ml each) for 2 h at room temperature followed by dialysis against PBS at 4°C, resulting in complete conjugation and no insoluble precipitate. The proportion of reduced sulfhydryl groups was quantitated prereduction, postreduction, and postconjugation using Ellman’s reagent (Pierce). All conjugates were stored at 4°C. Completeness of Id conjugation to KLH after glutaraldehyde and maleimide treatments was verified using nonreducing SDS-PAGE, showing no free Id.


Figure 1
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  		FIGURE 1. Comparison of glutaraldehyde and maleimide tumor Ag-carrier protein conjugation chemistries. A, Glutaraldehyde cross-links proteins primarily via the {epsilon}-amino groups of lysine residues, resulting in an irreversible amide bond between Ag (Id) and carrier protein (KLH). B, The maleimide group of the sulfo-SMCC linker in maleimide-activated KLH reacts with reduced sulfhydryl groups on the Ig to form a stable thioether bond.

 
Tumor challenges

Mice were injected with 1 x 105 A20 or 5 x 103 38C13 tumor cells s.c. on day 0 for early established tumor models or 1 wk after the third vaccination for prophylactic tumor challenge models. Mice were sacrificed when tumors reached 1.4 cm in diameter per institutional guidelines.

Therapeutic and prophylactic vaccinations

Groups of 8–12 mice were vaccinated with glutaraldehyde or maleimide Id-KLH conjugates (100 µg total: 50 µg of Id plus 50 µg of KLH) s.c. on days 4, 11, and 18 for early established therapeutic tumor models and days –21, –14, and –7 for prophylactic tumor challenge models. GM-CSF (55 ng) was coinjected with each Id-KLH vaccine at the identical site daily for 3 consecutive days as an adjuvant (11).

Treatment of early established BCL-1 tumors

Tumor cells were obtained from the spleens of premorbid mice bearing BCL-1 tumors and cryopreserved until use. Mice were inoculated i.p. with 1 x 105 BCL-1 tumor cells on day 0, and on day 4 began three weekly immunizations with maleimide or glutaraldehyde Id-KLH conjugates plus GM-CSF as described above or HBSS as a control, and survival was monitored. Mice bearing BCL-1 tumors were sacrificed when they reached a moribund state according to institutional guidelines.

Quantitation of anti-Id Abs by ELISA

Vaccinated mice were bled 4 days before tumor challenge. For A20 Id, serum was added to 96-well Nunc-Immuno Maxisorp plates (Nalge Nunc) coated with purified F(ab')2 A20 Id protein (3D6.3) and serially diluted. F(ab')2 A20 Id was generated using the F(ab')2 preparation kit (Pierce). Anti-Id Abs were detected using HRP-conjugated anti-mouse IgG ({gamma} specific; Southern Biotech). The 1G6 anti-A20 Id Ab was used as a standard. For IgM-expressing tumors, serum was added to plates coated with 38C13 or BCL-1 Id proteins. The S1C5 anti-38C13 Id Ab was used as a standard for 38C13. For BCL-1, serum dilution curves were used to evaluate anti-Id levels. Anti-Id isotype levels were evaluated using HRP-conjugated anti-mouse IgG1 (Southern Biotech), IgG2a (Southern Biotech), and IgG2b (Caltag Laboratories) Abs. Absorbance was determined with ABTS substrate at 405 nm using a SpectraMax Plus 384 microplate reader (Molecular Devices).

Surface staining of tumor cells with Id-KLH immune sera

Tumor cells were pretreated with Fc receptor-blocking anti-CD16/CD32 Ab (clone 2.4G2; BD Biosciences) and then incubated with a 1/10 dilution of naive or immune sera. Anti-Id Abs bound to A20 were detected with anti-IgG1-FITC (LO-MG1; Caltag Laboratories) or anti-IgG2b-FITC (LO-MG2b; Caltag Laboratories). Anti-Id Abs bound to 38C13 and BCL-1 were detected with anti-IgG-FITC (Caltag Laboratories). Cells were analyzed using a BD FACScan flow cytometer (BD Biosciences) with FACS Express software (De Novo Software).

Passive administration of immune sera

BALB/c mice were vaccinated with A20 maleimide Id-KLH on days 0, 14, and 28 (plus GM-CSF as above), and sera were collected on day 38. Naive mice were challenged with 1 x 105 A20 tumor cells i.p. and 8 h later were injected i.p. with 3 mg of A20 anti-Id IgG (determined by ELISA) in 450 µl of immune sera or an equal volume of naive sera or HBSS, and followed for survival.

T cell depletions

T cell subsets were depleted prevaccination as previously described (12, 39, 42). Depletions were validated by flow cytometric analysis of blood collected from three representative mice per group on day –1 (>99% depletion; data not shown).

B cell depletion

Mice were injected with 250 µg of the anti-murine CD20 mAb 18B12-2a (IgG2a; Biogen-Idec) (43) or control mouse Ig (UPC-10) i.v. on days –14, –7, 0, and weekly thereafter. B cell depletions (>95%, data not shown) were validated by flow cytometric analysis of blood, lymph nodes, spleen, and peritoneal lavage collected from 1 mouse per group on day –1 using anti-CD19-PE (clone 1D3; BD Biosciences). Anti-Id titers were measured in all mice by ELISA as described above to further confirm the functional depletion of B cells.

Ig cleavage from maleimide carrier protein conjugates

Lysosomes were isolated from mouse splenocytes using the lysosomal isolation kit (Sigma-Aldrich). Conjugates were subjected to lysosomal processing conditions (5–10 µg of microsomal proteins in 15 µl of 50 mM sodium citrate buffer (pH 4.5) containing 0.5% Triton X-100 and 2 mM DTT, for 1 h at room temperature) as previously described (44). OVA-BSA and OVA-A20 Id conjugates were prepared using maleimide-activated OVA (Pierce) or glutaraldehyde as described for Id-KLH conjugations. Treated conjugates, KLH, OVA, BSA, and free Id controls were analyzed by nonreducing SDS-PAGE and stained with Coomassie brilliant blue.

Conjugation of human Igs to KLH using maleimide

Mariculture KLH (Pierce) was activated with sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC; Pierce) as per the manufacturer’s instructions. Excess cross-linker was removed with a Zeba desalt spin column (Pierce) and the protein was equilibrated in 1x PBS with 100 mM EDTA. Human Igs (monoclonal IgG1, monoclonal IgG3, and polyclonal IgG (Sigma-Aldrich) in PBS were first reduced in 0.1 mM DTT (Sigma-Aldrich) for 1 h at 37°C, dialyzed into 1x PBS containing 0.1 M EDTA, and then conjugated at a 1:1 (w/w) ratio of Ig to KLH as per the manufacturer’s instructions. Ellman’s reagent was used to determine the relative amounts of free sulfhydryl groups in the Igs prereduction, postreduction, and postconjugation. Using the chosen conditions, approximately half of the total cysteine residues present in human Ig proteins were reduced before KLH cross-linking (data not shown).

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). p values were considered statistically significant at p < 0.05. Ab titer data was compared using the paired, two-tailed Student t test, and differences were considered statistically significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Vaccination with maleimide-conjugated Id-KLH provides superior eradication of A20 lymphoma compared with a traditional glutaraldehyde Id-KLH conjugate

A model of early established A20 B cell lymphoma was chosen to compare maleimide-conjugated Id-KLH to standard glutaraldehyde Id-KLH. We first established a specific set of conditions for achieving sulfhydryl-based conjugation of A20 Id to KLH. This involved partial reduction of Id with DTT, immediate transfer to EDTA-containing buffer to preserve reduced sulfhydryl groups, and subsequent reaction with maleimide-activated KLH. This resulted in complete conjugation to KLH, with no free Id detectable by SDS-PAGE and no insoluble precipitate (data not shown). To test the in vivo activity of the conjugates, mice were given a lethal s.c. inoculum of A20 tumor cells and 4 days later began a series of 3 weekly s.c. immunizations with Id-KLH plus GM-CSF (Fig. 2A). As reported previously, glutaraldehyde A20 Id-KLH conjugates induced no significant protection compared with HBSS control (p = 0.14) (28, 29). In contrast, maleimide Id-KLH vaccination eradicated tumors from the majority of mice (58%), with survival significantly superior to HBSS control or glutaraldehyde-conjugated Id-KLH (p < 0.0001 and p = 0.0004, respectively) (Fig. 2A). The specificity of the maleimide A20 Id-KLH antitumor response was demonstrated by comparison to vaccination with an irrelevant IgG2a (UPC10) conjugated to KLH via maleimide. Mice vaccinated with the A20 Id-KLH maleimide conjugate showed superior survival (75%) compared with irrelevant UPC10-KLH conjugate (0%, p = 0.0002) (data not shown). GM-CSF was critical to attaining antitumor immunity in this model, as vaccination with A20 Id-KLH conjugates without GM-CSF had slightly extended survival but no significant tumor protection compared with HBSS using either the maleimide or glutaraldehyde methods (p = 0.094 and p = 0.083, respectively) (data not shown). Fig. 2B illustrates that the prereduction of Id before glutaraldehyde conjugation to KLH did not enhance the level of tumor eradication compared with standard glutaraldehyde conjugation (both 25%) (p = 0.93). Maleimide Id-KLH once again resulted in significant tumor eradication (66.7%) compared with both reduced and nonreduced glutaraldehyde conjugates (p = 0.010 and 0.011, respectively). Given the difference in the two conjugation chemistries, we next asked whether each of the conjugates might preserve different epitopes for an anti-Id immune response. Maleimide Id-KLH alone and combination with an equal amount of glutaraldehyde Id-KLH elicited equivalent levels of tumor eradication (58.3%, Fig. 2C). Therefore, maleimide Id-KLH was sufficient to achieve maximal antitumor effects. Notably, the addition of glutaraldehyde conjugate did not appear to enhance or decrease the immunogenicity contributed by maleimide Id-KLH.


Figure 2
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  		FIGURE 2. Id protein conjugated to KLH via a maleimide-sulfhydryl linkage elicits superior tumor eradication compared with glutaraldehyde Id-KLH conjugates. A, Mice were inoculated s.c. with A20 tumor on day 0 and on day 4 began three weekly s.c. immunizations with maleimide or glutaraldehyde Id-KLH conjugates plus GM-CSF, or HBSS as control, and were followed for survival. Tumor was eradicated from the majority of mice receiving maleimide Id-KLH (p < 0.0001 vs HBSS), whereas no significant protection was afforded by glutaraldehyde Id-KLH. B, Prereduction of Id protein does not account for the superior protection seen with maleimide Id-KLH over glutaraldehyde conjugates. Prereduced (as with maleimide) or native Id proteins were conjugated to KLH using glutaraldehyde and used to immunize mice as described above. C, The combination of maleimide and glutaraldehyde Id-KLH conjugates does not enhance or diminish antitumor immunity above that of maleimide Id-KLH alone. Immunization with a combination of 200 µg of Id-KLH protein (100 µg of glutaraldehyde Id-KLH plus 100 µg of maleimide Id-KLH) was compared with 100 µg of each Id-KLH protein complex alone.

 
A20 maleimide Id-KLH conjugate vaccination results in both T cell and humoral responses, with CD8+ T cells being the critical effector mechanism for tumor eradication

Prophylactic (vaccination followed by tumor challenge) experiments were conducted to allow the measurement of anti-Id Abs without clearance from the circulation by binding to growing tumor cells or free Id protein released from the tumor (6). Both maleimide and glutaraldehyde Id-KLH induced significant tumor protection compared with HBSS controls (p < 0.0001) (65 vs 25%, respectively) (Fig. 3A), although protection after maleimide Id-KLH was superior (p = 0.0073). Unconjugated A20 Id did not elicit protective immunity (data not shown). Although both maleimide and glutaraldehyde Id-KLH conjugates induced anti-Id Abs (Fig. 3B), maleimide Id-KLH generated far higher anti-Id levels (p < 0.0001). Anti-Id Abs in maleimide Id-KLH immune sera were almost exclusively of the IgG1 isotype (with trace amounts of IgG2a and IgG2b) by both ELISA (data not shown) and flow cytometry measuring binding to the surface of A20 tumor cells (Fig. 3C). To determine the role of anti-Id Abs vs T cells in the protective immunity against A20, tumor inoculation was followed by a large dose of anti-Id immune sera derived from mice vaccinated with maleimide Id-KLH. Transfer of immune sera did not confer protection from tumors compared with naive sera or HBSS (Fig. 3D). In contrast, A20-bearing mice depleted of CD8+ or CD4+ T cell subsets before vaccination demonstrated a critical role for CD8+ T cells in tumor eradication (Fig. 3E). Mice depleted of CD8+ T cells succumbed to tumor as quickly as HBSS controls (p = 0.26), while mice depleted of CD4+ T cells survived as well as those given control Ab (p < 0.0001 vs HBSS).


Figure 3
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  		FIGURE 3. Maleimide Id-KLH conjugate vaccination in the A20 model results in both T cell and humoral responses, with CD8+ T cells being the dominant effector mechanism for tumor eradication. A, Maleimide Id-KLH provides superior protective antitumor immunity over glutaraldehyde Id-KLH. Mice received three weekly immunizations with maleimide or glutaraldehyde Id-KLH conjugates plus GM-CSF, or HBSS, and were challenged 7 days later with s.c. tumor on day 0 and followed for survival. Data are pooled from two replicate experiments (n = 20 mice/group). B, Maleimide Id-KLH conjugates elicit superior anti-Id Ab titers. Anti-Id Ab levels in the serum of the mice vaccinated in A were determined by ELISA in serum collected 4 days before tumor challenge. **, p < 0.0001 compared with glutaraldehyde Id-KLH conjugates anti-Id titers. C, Anti-Id Abs induced by maleimide Id-KLH conjugates recognize Id on the surface of tumor cells. Naive and immune sera collected from the mice treated in A were used to stain A20 tumor cells, and bound IgG1 and IgG2b anti-Id Abs were measured by flow cytometry. Black lines represent specific Ab staining, dark gray lines are isotype controls, and light gray histograms represent unstained cells. D, Passive transfer of high-titer maleimide A20 Id-KLH immune serum does not protect against A20 lymphoma. BALB/c mice were injected i.p. with tumor cells followed by injection with immune sera from maleimide Id-KLH-vaccinated mice containing 3 mg of A20 anti-Id Abs, naive sera, or HBSS. E, CD8+ T cells are critical for tumor eradication in mice vaccinated with A20 maleimide Id-KLH. BALB/c mice were depleted of CD4+ and CD8+ T cells starting on day –6, injected with A20 tumor on day 0, and 4 days later began three weekly immunizations with maleimide Id-KLH.

 
Maleimide Id-KLH conjugates induce superior anti-Id immunity in the 38C13 lymphoma model

The 38C13 B cell lymphoma has served as the prototypical model in establishing Id-KLH vaccination strategies for human trials (6, 7, 9, 10, 11). Vaccination with 38C13 glutaraldehyde Id-KLH can generate protective tumor immunity mediated chiefly by anti-Id Abs (7, 10, 12). Mice were given three weekly vaccinations of maleimide or glutaraldehyde Id-KLH and challenged with tumors. As in the A20 model, maleimide Id-KLH was significantly more potent than glutaraldehyde Id-KLH in eliciting tumor protection (85 vs 47%, respectively, p = 0.024; Fig. 4A). The improved survival seen in maleimide Id-KLH-treated mice was associated with significantly higher anti-Id Ab levels compared with those in glutaraldehyde Id-KLH-treated mice (161 ± 54 µg/ml vs 52 ± 23 µg/ml, respectively, p < 0.0001) (Fig. 4B). By both ELISA and flow cytometry these anti-Id Abs were largely of the IgG1 isotype, but significant amounts of IgG2a were also detected (maleimide, 31.6 ± 12.5 µg/ml; glutaraldehyde, 13.1 ± 1.1 µg/ml). Anti-Id Abs of the IgG2a isotype are known to be more effective than IgG1 against 38C13 lymphoma (12, 45).


Figure 4
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  		FIGURE 4. 38C13 maleimide Id-KLH conjugate vaccines induce superior antitumor protection and higher anti-Id Ab titers compared with glutaraldehyde Id-KLH. A, Tumor protection after three weekly maleimide or glutaraldehyde Id-KLH vaccinations followed by 38C13 tumor challenge 7 days later. Data were pooled from two replicate experiments (n = 20 mice/group). B, Maleimide Id-KLH elicits significantly higher anti-Id Ab levels than glutaraldehyde Id-KLH. Serum anti-Id Ab levels in mice vaccinated with Id-KLH were determined 4 days before tumor challenge. C, CD4+ T cells are critical for antitumor immunity against 38C13. Mice were depleted of CD4+ or CD8+ T cells using mAbs before Id-KLH vaccination and subsequent tumor challenge. D, B cells are required for protective immunity against the 38C13 tumor. Mice were injected with B cell-depleting or control Abs before Id-KLH vaccination and subsequent tumor challenge. E, B cell depletion results in complete impairment of humoral anti-Id immunity, which correlates with overall survival. Anti-Id titers were determined after three vaccinations 1 day before tumor challenge. **, p < 0.0001 compared with controls.

 
Mechanisms of antitumor immunity after vaccination with 38C13 maleimide Id-KLH

Depletion of CD8+ T cells before maleimide Id-KLH vaccination did not diminish 38C13 tumor protection, as survival was comparable to that of control mice (p = 0.98) (Fig. 4C). However, mice predepleted of CD4+ T cells died nearly as fast as controls, indicating an essential role for CD4+ T cells in tumor protection. This likely reflects a requirement for T cell help in generating anti-Id Abs rather than a direct MHC-restricted cytotoxic effector function, as 38C13 lacks class II MHC expression. To confirm the role of humoral immunity in this model, a mouse anti-CD20 mAb was administered to deplete B cells before vaccination (43). 38C13 lymphoma cells do not express CD20 (data not shown) and, therefore, the depleting Ab would only affect the endogenous B cell compartment. Anti-CD20 treatment resulted in >95% depletion of normal B cells (data not shown) and completely abrogated tumor protection after maleimide Id-KLH vaccination (p < 0.004 compared with controls, Fig. 4D). No statistically significant difference in tumor protection was seen comparing maleimide Id-KLH with or without control Ab treatment. Anti-CD20-treatment also eliminated the generation of anti-Id Abs in vaccinated mice, while mice given maleimide Id-KLH with or without control Ab developed high titers of protective Abs (p < 0.0001) (Fig. 4E). Thus, anti-Id Abs represent the dominant mechanism of 38C13 tumor protection after maleimide Id-KLH vaccination, as previously shown for glutaraldehyde Id-KLH (7, 10, 12).

Maleimide Id-KLH conjugates achieve superior tumor eradication in the BCL-1 lymphoma model

A third B cell lymphoma model, BCL-1, also used in prior studies of experimental Id vaccination (8, 39, 46), served to further validate the maleimide Id-KLH conjugation method. Early established BCL-1 tumors were successfully eradicated from 50% of mice using maleimide Id-KLH (p = 0.018 vs HBSS control), while the glutaraldehyde conjugate did not result in statistically significant protection compared with controls (p = 0.28) (Fig. 5). Maleimide BCL-1 Id-KLH immunization also induced 3-fold higher levels of anti-Id Abs than glutaraldehyde conjugate (p < 0.0001) (data not shown).


Figure 5
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  		FIGURE 5. Vaccination with maleimide Id-KLH conjugate successfully eradicates early established BCL-1 murine B cell lymphoma. Mice were inoculated i.p. with BCL-1 tumor on day 0 and on day 4 began three weekly immunizations with maleimide or glutaraldehyde Id-KLH conjugates plus GM-CSF, or HBSS as control, and followed for survival.

 
The extent of glutaraldehyde but not maleimide Id-KLH conjugation significantly affects the potency of the antitumor immune response

In current clinical trials, glutaraldehyde conjugation is continued for 1–2 h regardless of the sensitivity of individual Id proteins to the cross-linking agent, which is dictated by the amino acid content of the individual tumor Id. We theorized that glutaraldehyde cross-linking, as it progressively cross-links not only lysine but also cysteine, tyrosine, and histidine residues, might impair the immunogenicity of an Id-KLH conjugate if the reaction proceeds past an optimal time point, leading to "overconjugation". To test this, 38C13 Id was conjugated to KLH with glutaraldehyde for varying lengths of time. Visible precipitates became apparent after 5–10 min of conjugation with glutaraldehyde, and longer incubation times resulted in greater amounts of insoluble precipitate. All conjugates were then evaluated for antitumor effects in vivo. The optimal duration of glutaraldehyde Id-KLH conjugation for 38C13 was 30 min (58% tumor protection), with lesser protection afforded by shorter (2 or 15 min.) or longer (60 or 120 min.) conjugations (Fig. 6, A and B). Still, maleimide Id-KLH elicited the highest proportion of long-term survivors (83%; Fig. 6, A and B), inducing significantly better protection than both the 2 min and 2 h glutaraldehyde conjugates (p < 0.031). Anti-Id Ab titers mirrored the survival results (Fig. 6C), with maleimide exceeding even the best (30 min.) glutaraldehyde conjugate. Thus, even an optimized glutaraldehyde Id-KLH vaccine could not match the antitumor efficacy of maleimide Id-KLH. Furthermore, the duration of maleimide conjugation did not affect the level of tumor protection, as vaccine efficacy remained high at all reaction durations (Fig. 6D). In addition, levels of induced anti-Id Abs remained uniformly high regardless of conjugation duration (Fig. 6E).


Figure 6
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  		FIGURE 6. The duration of glutaraldehyde, but not maleimide, conjugation critically affects the subsequent antitumor immune response against 38C13 lymphoma. A and B, Degree of glutaraldehyde conjugation affects tumor-protective capacity of an Id-KLH vaccine. Mice were vaccinated with 38C13 Id conjugated to KLH with glutaraldehyde for varying lengths of time or with standard maleimide Id-KLH, challenged 7 days later with tumor and followed for survival. *, p < 0.031 compared with maleimide Id-KLH conjugates. C, Optimal anti-Id Ab titers elicited by glutaraldehyde conjugates are highly dependent on the duration of conjugation. Anti-Id Ab levels in the serum of mice in A and B were determined by ELISA 4 days before tumor challenge. **, p < 0.0005 compared with maleimide Id-KLH conjugates. D, Maleimide Id-KLH conjugates elicit optimal tumor protection regardless of the length of conjugation. 38C13 Id was conjugated to KLH using maleimide-activated KLH for various time intervals, and mice were vaccinated as above. E, Mice vaccinated with maleimide Id-KLH attain high levels of anti-Id Ab levels regardless of the duration of time the conjugation reaction was allowed to proceed.

 
Id is cleaved from maleimide, but not glutaraldehyde, Id-KLH conjugates under physiologic lysosomal processing conditions

Tumor Ag proteins require uptake by APCs and proteolytic processing in lysosomes to elicit tumor-specific T cell responses (47). Thus, an optimal Id-KLH vaccine would allow release of Id upon uptake by APCs and minimize damage to amino acid residues within the tumor Ag. Glutaraldehyde, in forming numerous protein-protein linkages, might impair Ag processing, whereas proteins linked via only reduced cysteine residues might be more susceptible to lysosomal proteolysis. To test this, maleimide and glutaraldehyde Id-KLH conjugates were incubated with lysosomal extracts in vitro under acidic lysosomal conditions (44) and subjected to nonreducing SDS-PAGE. Fig. 7A illustrates that Id could not be released from glutaraldehyde Id-KLH under these conditions. High molecular mass complexes remained at the top of the gel, as did glutaraldehyde KLH-KLH. In contrast, Ig heavy and light chains were efficiently cleaved from maleimide Id-KLH, indicating the ability of acidic lysosomal conditions to release tumor Ag for processing and presentation. Released Id dissociated into heavy and light chains under these lysosomal conditions; however, SDS-PAGE revealed no further protein degradation, possibly due to the short incubation time. In subsequent experiments, we found that among the individual components of the lysosomal conditions, either pH 4.5 or physiologic reducing conditions were sufficient to break the Id-KLH linkage while enzymes alone had no such effect (data not shown).


Figure 7
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  		FIGURE 7. Cleavage of protein-carrier SMCC linkage, but not glutaraldehyde protein-carrier linkage, under lysosomal processing conditions. Nonreducing SDS-PAGE analysis of maleimide or glutaraldehyde protein conjugates with or without exposure to in vitro lysosomal conditions. A, A20 Id or various KLH conjugates after preincubation in lysosomal conditions for 60 min. Proteins were loaded with equal concentrations of Id (10 µg) represented in each Id-containing sample. Id is not released from glutaraldehyde Id-KLH, as high molecular mass complexes remain at the top of the gel, just as with the KLH-KLH glutaraldehyde conjugate. B, Complete cleavage of a maleimide BSA-OVA conjugate under lysosomal conditions. OVA, BSA, or BSA-OVA conjugates with or without 60 min of pretreatment in lysosomal conditions. Proteins were loaded with equivalent concentrations of OVA and/or BSA represented in each sample. C, Complete cleavage of Id from a maleimide carrier protein conjugate under lysosomal conditions. OVA, A20 Id, or maleimide Id-OVA conjugate with or without 60 min pretreatment in lysosomal conditions are shown. Proteins were loaded with equivalent concentrations of OVA and/or A20 Id represented in each sample.

 
Due to the high molecular mass of KLH (>400 kDa) and the inability to resolve free vs Id-bound KLH, these results do not distinguish cleavage of the succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) Id-KLH linker from the cleavage of interchain disulfide bonds. However, approximately equal amounts of free Id were seen in the maleimide Id-KLH and Id alone lanes, indicating that most Id was released from maleimide Id-KLH. To confirm that the maleimide Ag-carrier protein linkage itself could be cleaved, we studied two smaller monomeric proteins linked via SMCC. Maleimide OVA-BSA and OVA-Id conjugates dissociated completely under acidic lysosomal conditions (Fig. 7, B and C), indicating cleavage of the SMCC linker, whereas glutaraldehyde conjugates remained linked. Thus, the release of Id from carrier protein under lysosomal conditions offers a potential mechanism for the enhanced immunogenicity of maleimide conjugates.

Conjugation of human immunoglobulins to KLH using maleimide

Current Id-KLH vaccines undergoing phase III clinical testing contain tumor-specific recombinant IgG1 or IgG3 or rescue hybrid-derived IgG or IgM (26). Fig. 8 illustrates complete conjugation of human IgGs to maleimide-activated KLH and the quantitative monitoring of free sulfhydryl content at various stages during conjugation. Thus, human Igs could be efficiently conjugated to KLH for the production of therapeutic Id-KLH vaccines.


Figure 8
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  		FIGURE 8. Demonstration of complete conjugation of human IgGs to KLH carrier protein using maleimide chemistry. A, Monitoring the level of free sulfhydryl groups during various stages of human IgG conjugation to KLH via maleimide conjugation using Ellman’s reagent. B, Human IgG1 and polyclonal IgG were conjugated 1:1 to maleimide-activated KLH. Conjugates were subjected to nonreducing SDS-PAGE alongside the corresponding free IgGs. No free Ig is seen in the IgG-KLH lanes, indicating that Ig is completely conjugated to the carrier protein and stable under nonreducing conditions.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Id vaccination has shown promising results in phase I/II trials for non-Hodgkin’s B cell lymphomas, and glutaraldehyde Id-KLH vaccines are now being formally evaluated for efficacy in several phase III randomized clinical trials (26). Nonetheless, current Id-KLH vaccines remain far from optimal, as they often fail to induce anti-Id immune responses, tumor regression, or prolonged survival. Using an alternative conjugation technique, we now demonstrate in several well-characterized murine lymphoma models a surprising level of efficacy compared with the traditional glutaraldehyde method currently viewed as standard after 20 years of investigational usage (6, 25). The maleimide method allowed one Id-KLH vaccine (A20 lymphoma), previously believed to be ineffective (28, 29), to have striking efficacy at eradicating early established tumors. Moreover, in the prototypical 38C13 and BCL-1 models, superior antitumor effects were also achieved with the new maleimide Id-KLH vaccine. Current clinical trials using glutaraldehyde to conjugate patient Id proteins to KLH may thus be limiting the potential effectiveness of the vaccines by suboptimal cross-linking of the proteins.

Several observations led us to question the use of glutaraldehyde as the preferred cross-linking agent for Id-KLH vaccines. First, the A20 murine B cell lymphoma was curiously reported to be unresponsive to glutaraldehyde Id-KLH vaccination (28, 29) while recombinant forms of A20 Id provided some tumor protection (28, 31, 48). This suggested that glutaraldehyde might be damaging one or more essential immunogenic epitopes during conjugation. Indeed, several class I MHC-binding nonapeptides within the A20 Id heavy chain have been identified (31), with tyrosine and lysine residues in or flanking critical anchor positions that could be susceptible to glutaraldehyde inactivation. Second, prolonged conjugation of murine or human Id proteins to KLH with glutaraldehyde can result in large, insoluble aggregates with presumed loss of important immunogenic epitopes. Incubation of Id and KLH proteins with glutaraldehyde can also lead to undesired Id-Id and KLH-KLH conjugates devoid of antitumor activity (6). In the original Id vaccine studies by Levy and colleagues at Stanford University, Id-KLH vaccines were prepared by hand with the duration of glutaraldehyde conjugation individually titrated until the initial formation of visible precipitates, and the reaction was then stopped in an attempt to achieve an optimal level of conjugation (13, 20). In contrast, current trials use a fixed 1 to 2 h conjugation period, which yields variable amounts of insoluble precipitated material (J. Timmerman, unpublished observations). Third, Id-specific immune response rates have varied from 48 to 80% in clinical trials of glutaraldehyde Id-KLH vaccines (16, 17, 18, 19, 20, 21, 22, 23, 24, 27), leaving many patients without detectable anti-Id immunity.

The heterobifunctional agent sulfo-SMCC was chosen to limit KLH conjugation only to free sulfhydryl groups in the Id (Fig. 1). It was hypothesized that maleimide Id-KLH conjugates would have increased immunogenicity due to the preservation of tumor Ag epitopes, thereby endowing an Id-KLH vaccine such as A20 with the capacity to elicit potent antitumor immunity. Indeed, a surprising level of efficacy was observed in a model of early established A20 lymphoma, with tumor eradication consistently more than twice that obtained using glutaraldehyde Id-KLH (Fig. 2). The murine lymphoma A20 could be analogous to those of many patients who do not mount immune or clinical responses to current glutaraldehyde Id-KLH vaccines, suggesting that use of maleimide conjugates in human Id vaccines might improve clinical response rates.

In lymphoma patients, both anti-Id Abs (16, 49) and T cells (15, 17, 20) can be important mediators of antitumor effects after Id-KLH vaccination. However, it is impossible to predict which of these effectors will be most critical to antitumor immunity in a given individual. Thus, the induction of strong Ab and T cell anti-Id immune responses should be an important goal of any Id vaccine strategy. Maleimide Id-KLH conjugates appear to fulfill this goal by stimulating superior anti-Id immunity in models dependent on either CD8+ T cells (A20) or Abs (38C13). In mice vaccinated with A20 maleimide Id-KLH, antitumor immunity appeared to be due primarily to CD8+ T cells rather than Abs, as demonstrated by in vivo T cell depletion studies and adoptive serum transfer (Fig. 3). These results are in agreement with our original supposition that glutaraldehyde cross-linking may impair the structure or processing of critical amino acid residues in or flanking the previously reported class I MHC-restricted Id-derived peptides (31).

Because each lymphoma patient’s tumor Id will express a unique set of immunologic epitopes, an optimal Id vaccine would be one in which the full collection of potential B cell and T cell epitopes represented within the tumor-specific Ig sequences is preserved for immune recognition. Presentation of peptides on class I and class II MHC molecules requires proteolytic cleavage of ingested protein within lysosomes of APCs and subsequent binding of short peptides to MHC molecules for display to the adaptive immune system (47). The irreversible protein-protein bonds resulting from extensive glutaraldehyde cross-linking would be expected to impair Ag processing and presentation. Indeed, we found that under in vitro lysosomal conditions (44), Id was not released from glutaraldehyde-conjugated Id-KLH (Fig. 7A). However, while maleimide Id-KLH was stable at neutral pH, Id could be released from the conjugate under lysosomal processing conditions, possibly allowing more efficient proteolytic processing of the Id for presentation on MHC molecules. These findings were surprising in that the linker resulting from SMCC/maleimide cross-linking is currently classified as noncleavable (see Crosslinker Selection Guide from Thermo Fisher Scientific at www.piercenet.com).

B cell epitopes also appear to be better preserved and presented by using maleimide vs glutaraldehyde conjugates. In all three models tested, maleimide Id-KLH induced significantly higher titers of anti-Id Abs (Figs. 3, 4, and 6). Importantly, the induced Abs could recognize Id as presented in its native conformation on the surface of tumor cells. Interestingly, although vaccination with A20 maleimide Id-KLH induced high titers of anti-Id Abs, tumor immunity in this model relied on CD8+ T cells rather than Abs. Nonetheless, an anti-A20 Id mAb derived from a mouse vaccinated with maleimide Id-KLH has been found to possess potent anti-proliferative effects against A20 lymphoma both in vitro and in vivo (J. Timmerman and R. Levy, unpublished observations). Thus, maleimide Id-KLH is capable of eliciting Abs with important functional properties, possibly through the presentation of epitopes that might have been otherwise destroyed by glutaraldehyde (Fig. 3B). The potential for glutaraldehyde conjugation to damage important B cell epitopes was demonstrated in time course conjugation studies (Fig. 6). In the 38C13 model, the immunogenicity and tumor-protective capacity of a glutaraldehyde Id-KLH vaccine peaked after 30 min of conjugation and declined thereafter. In striking contrast, the maleimide Id-KLH conjugate retained its superior antitumor effect (83% protection) and the ability to induce anti-Id Abs even after a 2-h conjugation. Thus, maleimide yields more uniformly potent Id-KLH vaccine products from individual tumor-specific Id proteins.

The use of sulfhydryl-based conjugation appears to mark a substantial improvement in the efficacy of Id-KLH vaccines. The present study represents the first systematic evaluation of maleimide-based vs glutaraldehyde-based tumor Ag-carrier protein conjugate vaccines in experimental lymphoma models. Although Kaminski and colleagues found that the 38C13 Id protein coupled to thyroglobulin carrier protein using sulfo-SMCC could provide tumor protection (6), maleimide-based Id-KLH conjugation was not performed and the enhanced immunogenicity of maleimide over glutaraldehyde Id conjugates was not appreciated. Glutaraldehyde Id-KLH conjugation has thus remained the standard in both experimental models and clinical trials (7, 10, 11, 13, 16, 17, 20, 23, 24). Meanwhile, a number of other investigators have sought to use maleimide chemistry to enhance the immunogenicity of tumor Ag-carrier protein conjugate vaccines. Livingston and colleagues have extensively characterized the immune response to a variety of lipid, carbohydrate, and peptide tumor Ags linked to KLH, in general finding an advantage of maleimide over glutaraldehyde conjugation for the induction of humoral responses (50, 51). However, enhanced T cell responses against short MUC1 tumor Ag-derived peptides are not detectable in mice or humans after immunization with maleimide-conjugated MUC1-KLH (50, 52). Moreover, with short peptide Ags, sulfhydryl-based conjugation has not always been found to improve humoral immunogenicity. Kirkley et al. found that an HIV peptide coupled to KLH via glutaraldehyde induced higher levels of Abs in mice than a maleimide conjugate (53). Interestingly, however, the peptide-specific T cell response was stronger after immunization with maleimide conjugate. Our current report is unique in finding that full-length protein tumor Ags coupled to KLH via maleimide offer superior in vivo efficacy in three different lymphoma models and that the enhanced tumor immunity can be mediated by either T cells or Abs.

Given the consistently superior results we have observed with maleimide-based conjugation, this improved method deserves testing in future Id-KLH vaccine clinical trials. Sulfhydryl-based conjugation could be easily substituted for the current glutaraldehyde method, as the reaction chemistries are well defined. Maleimide-based linkers such as sulfo-SMCC have already been used in the manufacture of immunotoxins administered in human clinical trials (54, 55). The success of a sulfhydryl-based Id-KLH conjugates over traditional glutaraldehyde conjugates in the current study suggests that this approach might be extended to other protein Ag vaccines, such as those targeting tumor-specific T cell receptors (56) or Ags associated with solid tumors (57) or infectious diseases.


   Acknowledgments
 
We thank Dr. Ronald Levy (Stanford University, Stanford, CA) for helpful comments and suggestions, and Drs. Sherie Morrison and Gang Zeng (University of California, Los Angeles, CA) for critical evaluation of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was funded by the Albert Yu and Mary Bechmann Foundation, which we thank. J.M.T. is the recipient of a Clinical Investigator Award from the Damon Runyon Cancer Research Foundation (CI-26-05). Back

2 Address correspondence and reprint requests to Dr. John M. Timmerman, Division of Hematology and Oncology, Center for Health Sciences, Room 42-121, 10833 LeConte Avenue, University of California, Los Angeles Medical Center, Los Angeles, CA, 90095-1678. E-mail address: jtimmerman{at}mednet.ucla.edu Back

3 Abbreviations used in this paper: KLH, keyhole limpet hemocyanin; SMCC, succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate; sulfo-SMCC, sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate. Back

Received for publication February 15, 2008. Accepted for publication July 9, 2008.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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