Background
B cell malignancies express a highly tumor-specific antigen, the variable (V) regions of the monoclonal immunoglobulin (Ig), which contain antigenic determinants called idiotopes collectively known as idiotype (Id). Protein Id vaccination has been pursued as a therapeutic approach to B cell malignancies over the last 20 years [
1]. Immunologic and clinical responses have been detected [
1], whereas demonstration of clinical benefit is so far limited to follicular lymphoma [
2,
3].
Because of the very nature of the antigen (Ag) (i.e., Id), large scale clinical application of protein Id vaccination is limited by the need to prepare a custom-made vaccine for each and every patient. Overcoming this problem, DNA vaccination holds promise to streamline tailor-made vaccine manufacture by circumventing the need for purification of Ig protein (or derivatives thereof), conjugation to carrier protein (e.g. KLH) and administration of adjuvants. However, although effective in rodents, DNA vaccination has met with limited success thus far in humans due to low potency of vaccines [
4]. The poor immunogenicity of DNA vaccination can be improved by several means such as improved vector design and efficient electroporation [
5]. Another strategy is based on the finding that targeting of Ag to antigen-presenting cells (APC) enhances immunogenicity, as shown for chemical antibody (Ab)-Ag conjugates [
6,
7] and Ab-Ag fusion proteins [
8,
9]. Thus, we [
10] and others [
11,
12] cloned DNA constructs encoding proteins that target Ag to APC. When such constructs were injected
s.c. or
i.m., combined with electroporation, transfected host cells secreted fusion proteins that targeted APC for enhanced immune responses [
10]. In our previous studies, we used homodimeric Ig-based vaccines (Vaccibodies, VB), each chain consisting of a targeting unit, a dimerization unit and an antigenic unit. The dimerization unit consists of a shortened hinge region from hIgG3 whereas the N-terminal targeting unit can consist of either single chain fragment variable (scFv) specific for surface molecules on APC such as mouse MHC class II [
10], mouse CD40 [
13], human TLR2 and CD14 [
14], or natural ligands such as the mouse chemokines CCL3 (mCCL3) and CCL5 (mCCL5) [
15] and human CCL3 [
16]. Depending on targeting strategy, such VB proteins had a 10–10,000 fold increased efficiency to stimulate CD4
+ T cells
in vitro in mice [
10,
13,
15,
16] and humans [
14,
16]. Moreover, DNA Vaccibodies elicited superior antibody and T cell responses in mice, as well as greatly enhanced tumor protection [
10,
13,
15,
16]. In a stepwise, translational endeavour, the first fully murine Vaccibodies [
10] have been extended to chimeric murine/human Vaccibodies, including tailor-made Vaccibodies for multiple myeloma patients [
17].
A complementary strategy to streamline clinical Id vaccination is to exploit the high similarity of Ig V regions expressed by molecularly identified subgroups of patients with B cell malignancies. For example, the molecular characterization of Hepatitis C Virus (HCV) related lymphomas showed that more than 70% of these cases expressed either IGKV3-20 or IGKV3-15 light chains [
18‐
20], with a high degree of homology between individual lymphomas. Moreover, IGHV1-69 is expressed as the partner of IGKV3-20 or IGKV3-15 in up to 70% of HCV-related lymphomas [
18,
20]. Such commonly expressed B cell receptors (BCR) are called stereotyped receptors. Stereotyped BCRs are found also in several non HCV-associated B cell malignancies, such as MALT lymphomas [
21‐
23] and Chronic Lymphocytic Leukemia (CLL) [
24‐
26]. The analysis of VH CDR3 in more than 7000 VH (IGHV-IGHD-IGHJ) sequences from patients with CLL has established that CLL comprises two distinct categories: one with stereotyped and the other with heterogeneous BCR, in an approximate ratio of 1:2 [
27]. Thus, it could be envisioned that a number of off-the-shelf Id vaccines for molecularly identifiable subgroups of patients could be developed, obviating the need to tailor-make Id-vaccines for every patient. Although it is not known whether these Ids are immunogenic in the majority of patients, such off-the-shelf Id vaccines could cover up to 30% of patients with selected B cell malignancies, thus affording substantial savings in time and costs associated with Id vaccine manufacture.
On these premises, we have here produced fully human chemokine-Id fusion DNA Vaccibodies which due to cross-species reactivity of chemokines could be tested as DNA vaccines in mice. Moreover, using a panel of CLL patients’ cells and a mouse model for HCV-associated B cell lymphomas we explored the possibility of inducing cross-reactive anti-Id antibody responses following immunization with VB expressing a stereotyped B cell receptor.
Methods
Patient material
Patients diagnosed with CLL (see Table
1) were seen at the Department of Haematology outpatient clinic, Oslo University Hospital, Rikshospitalet, Oslo, Norway. Blood samples from 5 patients were procured following written informed consent using protocols approved by the Regional Committee for Medical and Research Ethics, South-East Norway. Blood samples were procured in tubes containing ACD as anticoagulant. Experiments were conducted on purified mononuclear blood cells.
Table 1
Characteristics of CLL patients’ BCR
103 | IgMλ | 3-48*02 | 2-2*01 | 4*01 | 3-21*01 | 3*02 |
106 | IgMλ | 3-23*01 | 4-23*01 | 4*02 | 3-21*01 | 1*01 |
107 | IgMκ | 3-30*02 | 4-17*01 | 5*02 | 4-1*01 | 4*01 |
111 | IgMλ - κ | 3-53*01 | n.a. | 1*01 | 3-21*01 λ | 3*02 |
3-20*01 κ | 1*01 |
116 | IgMκ | 4-59*01 | 2-15*01 | 2*01 | 3-20*01 | 1*01 |
Flow cytometry
Cells were stained with primary reagents and appropriate secondary reagents or control as indicated. The following biotinylated mAbs were used: anti human IgG (HP6017, Zymed), anti mouse IgD (TIB149, ATCC), anti mouse Ck (clone 187.1), anti mouse IgG1
a (clone 10.9, BD Pharmingen), anti mouse IgG2a
a (clone 8.3, BD Pharmingen), anti mouse IgG2a
b (clone 5.7, BD Pharmingen). Quantification of surface antigen on CLL cells was performed using mouse mAbs targeting human λ (clone 4C2) and human κ L chains (clone A8B5), and human IgM (clone 1030) from Diatec, Oslo, Norway, and the bead based Cellquant Calibrator kit (BioCytex, Marseille, France) according to the manufacturer’s guidelines [
28]. Cells (20,000) were acquired on a FacsCalibur (BD). Flow cytometry files were analyzed on CellQuest (BD) and Weasel v3.0 (
http://www.wehi.edu.au).
Mice and cell lines
BALB/c mice were obtained from Taconic (Ry, Denmark). B10.D2.C-TCRα
a/Bo (H-2
d, Ig haplotype IgH-C
b) mice were bred in house. These congenic mice are identical to B10.D2 except being congenic for the TCRα
a region [
29]. The studies were approved by the National Committee for Animal Experiments (Oslo, Norway). HEK 293E cells were from ATCC. The murine lymphoma Esb/MP cells [
30] were kindly provided by Jo Van Damme (Leuven, Belgium). The human Burkitt’s lymphomas DG-75 [
31] and PA682 [
32] were obtained from ATCC and kindly provided by Keith Thompson (Oslo, Norway), respectively.
Identification of V
H
and V
L
genes from CLL patients and assembly of scFv
V
H and V
L tumor-specific genes were cloned from preparations of cDNA from PBMCs of each patient. Tumor-specific transcripts were identified by PCR as previously described [
33].
Assembly of stereotyped scFv for Hepatitis C virus-associated B cell lymphomas
Plasmids encoding V (D) J
H (VH1-69, VD3-22, VJ4) and VJ
κ (VK3-20, VJ1) sequences from patients 1 and 2, respectively, were kindly provided by V. De Re (Aviano, Italy) and assembled into scFv as described above. This association has been shown to be representative of stereotyped Id molecules expressed by up to 70% of B cell malignancies associated with HCV chronic infection [
18,
20].
Assembly of patient-specific and cross-reactive Vaccibodies
The resulting scFv from individual CLL patients or the prototypic stereotyped BCR from HCV-associated B lymphoma were cloned C-terminal as antigenic units into the previously described VB format [
10]. In Vaccibodies used in this study, the N-terminal targeting unit consisted either of a mouse scFv from the 14-4-4S mAb specific for I-E mouse MHC class II molecules [
10], or the human chemokine LD78β (CCL3-L1) which binds CCR1, CCR3 and CCR5 [
16]. Control non-targeted Vaccibodies encoded a mouse scFv specific for the hapten NIP (5-iodo-4-hydroxy-3-nitrophenylacetyl), an antigen which is not found in the mouse tissue [
10]. The dimerization unit consisted of a shortened hinge (h1 + h4) and C
H3 of human IgG3 [
10].
Vaccine protein production and assessment of targeting properties
Expression and function of chimeric VB constructs was determined on supernatants from transiently transfected HEK 293E cells. To comparatively measure concentration of Vaccibodies expressing different targeting and antigenic units, an ELISA detecting the presence of human IgG3C
H3 in the dimerization unit was set up: mAb MCA878G (binds human IgG3C
H3, AB Serotec) as coat and biotinylated mAb HP6017 (binds to a different epitope in human IgG3C
H3) for detection. Binding to MHC class II was verified by admixing I-E
d-specific VB-containing supernatants and BALB/c (I-E
d+) A20 B lymphoma cells. Bound VB proteins were detected as previously described [
17]. NIP-specific Vaccibodies were tested for their ability to bind to NIP-BSA (conjugated in-house) as previously described [
17]. Chemotactic activity of LD78β (CCL3-L1) Vaccibodies on the mouse Esb-MP T cell lymphoma was tested by a transwell plate (Corning), as previously described [
16]. The results (mean + SE of duplicate samples) are presented as chemotactic index, defined as the fold increase in the number of migrating cells in the presence of chemotactic factors over the spontaneous cell migration (i.e., in the presence of medium alone).
Purification of patient tumor Ig protein
Heterohybridomas secreting tumor-specific Ig were generated from PBMCs of two patients with CLL by standard procedures [
35]. Hybridomas were screened by ELISA for expression of an Ig of expected H and L-chain isotypes. H-chain of hybridomas was verified by V
H sequencing. Hybridomas with V
H sequence fully matching that retrieved from patients’ CLL cells were selected for further study.
Generation of mouse B lymphoma cells (A20) that express a stereotyped BCR of HCV-associated B cell lymphomas
V (D) J
H regions from patient 1 (V
HP1) and VJ
L regions from patient 2 (V
κP2) were cloned into independent vectors that had been developed for membrane expression (but not secretion) of mouse IgDκ [
36]. In particular, V
HP1mIgDpLNOK vector expressing G418 resistance contains downstream of V
HP1 the murine germline IgD sequence (IgD
a allotype) in which the 3’ secretory exons had been eliminated, whereas V
κP2 was cloned in the pMUSmCκ expressing mouse constant κ region and zeocin resistance (Tuva Hereng and Bjarne Bogen, unpublished) to generate V
κP2MUSKAP.
A20 BALB/c B lymphoma cells that express an endogenous IgG2aκ and MHC class II (including I-Ed) were transfected with either or both vectors by electroporation, grown in selection medium (G418 and/or zeocin), and cloned by limiting dilution. To screen for A20 transfectants expressing patient VH, cells were stained with anti-IgD TIB149 (ATCC). Transfectants were selected by flow sorting for high IgD expression followed by cell culture and re-sorting. Expression of VH1-69 and VK3-20 was assessed by RT-PCR using the following primers: for VH1-69, forward GTGCAGCTGGTGCAGTCT and reverse TCCCTGGCCCCAATAGAAGT; for VK3-20 forward TTGTGTTGACGCAGTCTCCAG and reverse TTGATTTCCACCTTGGTCCCT.
As a negative control, we used an A20 cell that expressed IgDκ with V regions derived from the unrelated syngeneic Ab2-1.4 hybridoma [
36].
Mouse immunization
VB plasmids were purified with Endofree® Plasmid Mega Kit (Qiagen). 25 μL solution of 0.5 mg/mL VB DNA in sterile 0.9% NaCl (total 25 μg per mouse) was injected intradermally in the lower back of mice, on both sides, followed by electroporation using Derma Vax™ (Cyto Pulse Sciences, MD, USA). Groups consisted of 3 to 7 mice.
Measurement of antibody responses and assessment of specificity of anti-scFv antibodies
Blood samples were obtained at different time-points from the leg vein of vaccinated mice. Sera were tested by ELISA for reactivity against the patients’ CLL-derived monoclonal IgM or control isotype-matched IgM, or human IgG3, as coat. Bound antibodies were detected by either biotinylated mAb 187.1 (detects murine Cκ), anti-mouse IgG1a (clone 10.9 BD Pharmingen) or anti-mouse IgG2aa (clone 8.3 BD Pharmingen). The endpoint titres were recorded as the final serum dilution giving a signal above a fixed concentration of alkaline phosphatase-conjugated goat anti-human IgM (Sigma) or biotinylated mouse anti-human IgG (HP6017).
Sera were also used to stain PBMC from different CLL patients. Bound mouse antibodies were detected with biotinylated mAb 187.1 followed by streptavidin PerCP. Surface Ig expression by CLL cells was assessed by CellQuant (Biocytex), measuring the average Ig surface number by analyzing 20,000 cells.
Sera from immunized BALB/c or B10.D2.C-TCRaa/Bo (H-2d IgH-Cb) mice were used to stain A20 transfectants expressing human IGHV1-69, or human IGKV3-20, or both. Following blocking with PBS with BSA and heat-inactivated rat serum, transfectants were admixed with sera. Bound mouse antibodies were detected with biotinylated anti-mouse IgG1a for BALB/c sera or anti-mouse IgG2ab for B10.D2.C-TCRaa sera.
Injection of transfected A20 cells in immunocompetent mice
BALB/c mice were injected s.c. with 3 × 106 parental A20 cells or with A20 cells stably transfected with either VH1-69, or VK3-20 or both, and followed up for tumor growth.
Statistical analysis
Analysis of variance and regression analysis were conducted on all treatment arms. Results are presented as comparison of slopes of the antibody response in different treatment arms across serum dilutions.
Discussion
Herein we describe experiments aimed at generating Id vaccines for therapeutic Id vaccination of groups of patients with B cell malignancies expressing stereotyped BCRs. First, we demonstrate the feasibility of constructing fully human Id Vaccibodies with maintenance of both Id epitopes and functionality of targeting units (i.e., human CCL3 chemokine LD78β that cross-react with mouse CCRs) [
16]. Similar to fully murine and chimeric mouse/human vaccibodies, targeting antigen delivery to APC by human chemokine resulted in augmented immune responses in mice as compared with non-targeted control DNA Vaccibodies [
16]. Since these vaccines are fully human and isoform LD78β of human CCL3 bind cells expressing
Rhesus macaque CCR5 [
16], they are suited for both preclinical immunogenicity and regulatory toxicology studies in view of clinical application. It may be anticipated that targeting scFv Id to APC by LD78β could result in increased anti-Id responses in patients. Furthermore, DNA vaccination combined with electroporation is already employed in clinical trials for melanoma and prostate cancer, with mild to moderate, reversible side effects [
39].
Second, we explore a complementary approach to further streamline clinical application of Id vaccine for B cell malignancies.
Antibodies elicited in mice by Id DNA Vaccibodies constructed for a CLL patient showed cross-reactivity with CLL cells from some other patients. The most plausible explanation is that a fraction of mouse antibodies recognized epitope(s) displayed by the VL and/or VL/VH of the cross-reactive CLLs, since the CLL cells expressed nearly identical IGLV3-21 and similar IGHV3 family genes. With the HCV-NHL construct, the induced antibodies bound the transfected A20 cells but failed to bind a Burkitt’s lymphoma expressing the correct Vκ but not the corresponding VH, indicating a dominant anti-VH response. Even so, a contribution of the Vκ was however detected as VK3-20+VH1-69+ transfectants stained brighter than VkEndogenous/VH1-69only cells (that express an endogenous Vκ), suggesting responses towards VL/VH combinatorial idiotypes. Taken together, it is suggested that immunization of mice with fully human targeted scFv Id DNA vaccines could elicit antibodies that may react with either VL, or VH, or VL + VH, the relative proportions differing from case to case.
The above observations suggest the possibility of constructing vaccines covering molecularly identified subgroups of patients with B cell malignancies. This idea is supported by evidence of Id cross-recognition by anti-Id mAb [
40] as well as cross-reactive responses observed in clinical trials [
41‐
45]. A subgroup-specific, “off-the-shelf” Id vaccine should elicit a cross-reactive immune response effective against unrelated B cell tumors expressing V regions of the same families, provided that the pattern of somatic mutations is similar between individual tumors. In this respect, the application of criteria developed for clustering stereotyped BCR as based on HCDR3 sequences only [
27] do not fully meet the need for identifying patients amenable to immunization with such vaccines, as immune responses following vaccination may be directed to determinants located elsewhere in the V regions [
45‐
47]. Hence, similarity across the whole V regions should be evaluated. In principle, staining of lymphoma sections or single cell suspensions obtained from biopsy with serum from mouse that had been immunized with the intended Id vaccine could be able to identify candidate patients.
Evidence of clinical benefit by Id vaccination has been obtained so far only upon immunization with whole Ig protein vaccine [
2,
3], thus displaying to the host immune system both tumor-specific V
H and V
L. Therefore, considering that IGHV1-69 is often the partner of IGKV3-20 in HCV-related NHLs [
18,
20], a prototypic BCR for a subset of HCV-associated NHL was cloned, inserted into VB format and used to DNA immunize mice. We chose IGHV and IGKV from unrelated lymphomas purposely considering the possible use of such Vaccibodies as off-the-shelf, subgroup-specific vaccines. In fact, while HCV-associated NHL express IGHV1-69 and IGKV3-20 proteins with high similarity in the framework regions, the presence of several differences in the amino acid sequence of the CDR regions makes it difficult to select a one-for-all IGHV-IGKV pair. On these grounds, we selected IGHV1-69 and IGKV3-20 proteins as prototypic on the basis of their representativity of the FR regions among HCV-associated lymphomas. Our results show that such an artificial targeted DNA Id vaccine elicits antibodies in mice that bind mouse B lymphoma cells transfected with the H and L chain genes composing the artificial BCR. Whether these antibodies bind human IGHV1-69/IGKV3-20 B lymphomas remains to be investigated pending sample availability.
From a translational standpoint, the possibility of using DNA vaccines encoding scFv with the potential to elicit cross-reactive immune responses is not restricted to the IGHV1-69/IGKV3-20 combination, as similar features of conserved V regions usage have been detected in other B cell malignancies. Thus, one can envision tailored Id vaccines for each major stereotyped subset identified. To estimate the number of patients with B-cell malignancies that could be immunized with off-the-shelf cross reactive vaccines, a large database including sequences of idiotypic VH and VL genes expressed by low grade B-NHL, autoimmunity-associated lymphoproliferations (e.g. HCV-related NHL, mixed cryoglobulinemia, Sjögren’s syndrome) and CLL is currently being set up with the aim of identifying subgroups of tumors characterized by the expression of molecularly correlated Id proteins on the basis of the degree of sequence conservation among patients (R Dolcetti, unpublished results). However, stereotyped BCR sequences appear to be disease-biased. In CLL, shared V regions are in most cases unmutated [
25,
26,
48,
49] whereas in other B cell tumors (e.g., HCV-associated lymphomas, MALT lymphomas) somatic mutations are more frequent [
18,
20,
21]. Therefore, from an immunological standpoint, the yet unanswered question as to whether the host immune system can recognize V region sequences in germline configuration following Id vaccination is of paramount relevance for the possibility of applying Id vaccines, whether individual or subgroup-specific [
50].
It should be stressed that certain human B cell malignancies have not been described to express stereotyped BCR. One example is multiple myeloma cells that carry high loads of somatic mutations in their V regions, consistent with an origin from post germinal center B cells [
51]. In a previous report, we demonstrated that mice DNA-immunized with hybrid mouse/human Vaccibodies expressing scFv of either of four myeloma patients induced anti-Id antibodies that bound the corresponding myeloma protein with little cross-reactivity despite the fact that the BCR of two patients used the same IGHV and IGHJ genes [
16]. Thus, in the case of multiple myeloma, V regions of monoclonal Ig express unique Ids with little cross-reactivity, at least as defined by antibodies elicited by APC-targeted DNA Id vaccines.
The discussion above has focused on antibodies elicited by APC-targeted DNA Id vaccines since anti-Id antibodies have been linked to anti-lymphoma activity both in passive and active immunotherapy [
52,
53]. However, MHC-restricted, Id-specific T cells have been shown to display anti-lymphoma activity and to eradicate B cell tumors [
43,
47,
54,
55]. scFv in the vaccine contains Id-sequences available for MHC presentation. T cell responses were not investigated in this study as they would have been directed against xenogeneic Ig sequences thus having no semblance to the clinical situation. Nevertheless, it can be speculated that subgroup-specific vaccines can elicit cross-reactive Id-specific T cell responses which may or may not be accompanied by cross-reactive humoral responses, as has recently been suggested with HCV-associated B cell lymphoma IGKV3-20 [
56], However, patients differ in polymorphic HLA molecules and are therefore expected to present different sequences of V regions of CLL/B lymphoma BCR on their HLA molecules, thus making the possibility of cross-reactive T cell responses less common as compared with antibody responses. Also, based on mouse studies, T cell tolerance to the CLL/B lymphoma BCR is likely to limit T cell responsiveness in humans to a greater extent than humoral responses.
Competing interests
BB and PAR are inventors of Vaccibody patent applications filed by their employer (Univeristy of Oslo and Oslo University Hospital). BB is head of the scientific panel of the company Vaccibody AS.
Authors’ contributions
PAR, BB and RD conceived of the study. PAR carried out the construction and in vitro characterization of the vaccines, performed the mouse immunization studies, and drafted the manuscript. AO and LAM contributed to the CLL analysis and experiments. GET provided CLL patient samples, V region sequencing and clinical information. RD provided the plasmids for HCV-associated NHL BCR Vaccibody and helped to draft the manuscript. BB participated in the study design and coordination and helped to draft the manuscript. PAR, LAM and BB wrote the final manuscript. All authors read and approved the final manuscript.