Idiotype vaccines produced with a non-cytopathic alphavirus self-amplifying RNA vector induce antitumor responses in a murine model of B-cell lymphoma.

A promising therapy for patients with B-cell lymphoma is based on vaccination with idiotype monoclonal antibodies (mAbs). Since idiotypes are different in each tumor, a personalized vaccine has to be produced for each patient. Expression of immunoglobulins with appropriate post-translational modifications for human use often requires the use of stable mammalian cells that can be scaled-up to reach the desired level of production. We have used a noncytopathic self-amplifying RNA vector derived from Semliki Forest virus (ncSFV) to generate BHK cell lines expressing murine follicular lymphoma-derived idiotype A20 mAb. ncSFV/BHK cell lines expressed approximately 2 mg/L/24 h of A20 mAb with proper quaternary structure and a glycosylation pattern similar to that of A20 mAb produced by hybridoma cells. A20 mAb purified from the supernatant of a ncSFV cell line, or from the hybridoma, was conjugated to keyhole limpet hemocyanin and used to immunize Balb/c mice by administration of four weekly doses of 25 µg of mAb. Both idiotype mAbs were able to induce a similar antitumor protection and longer survival compared to non-immunized mice. These results indicate that the ncSFV RNA vector could represent a quick and efficient system to produce patient-specific idiotypes with potential application as lymphoma vaccines.

The Impact of the Hybridoma Technology on the R&D of Idiotypic Antibodies.

The PubMed data set was scanned with the title and abstract term "Idiotype" followed by secondary searches with "Vaccine" and "Clinical trial." The retrieved references were analyzed from the period before and after hybridoma technology (1975). In 1963, Oudin and Kunkel discovered that antibodies against antibodies can be raised to identify determinants unique to an antibody termed idiotype or individual antigenic determinant. Two laboratories reported that anti-idiotypic antibodies can suppress specific antibody responses in mice. In 1974, Jerne proposed a network of idiotypes and anti-idiotypes and the functionality of the idiotype network was confirmed. This prompted the proposal of a symmetrical regulatory immune response. By 1989, the concept and the functional parameters of the immune idiotype network were established in the prehybridoma period. It was not until 1981 that monoclonal anti-idiotypic antibodies were used as tools to study the expression of idiotypic determinants on antibodies and to categorize functional properties in the immune network as network antigens in 1989. Hybridoma-generated monoclonal anti-idiotypic antibodies provided the tools to precisely identify different idiotypic regions on antibodies and test these as targets to induce network cascades. The initial distinction of Ab2s as alpha and beta were expanded to include gamma and delta. The initial concept of Ab2beta being an antigen internal image, used as vaccine, was challenged showing that targeting all idiotopes on B cell receptors can induce specific antibodies. After the discovery of the hybridoma technology a wave of idiotype topic publications occurred, that declined by 2015. In 1985, in this wave of reports on anti-idiotypes, their importance to vaccines dominated. These vaccines targeted in animal models parasite, bacterial, and viral diseases, and cancer. The reported data indicated a therapeutic response in inbred mice. The issue of idiotype matching between mouse haplotypes of vaccine origin and treated mice were raised. In 1995, the human clinical trials in different cancers using anti-Id vaccines were reported. Only one such vaccine received conditional approval in Argentina and Cuba, whereas the other trials failed in phase II and III. The reasons for this failure were subsequently discussed. Although the use of the Milstein and Kohler hybridoma technology and subsequently alternative methods to produce monoclonal animal and human antibodies created a new class of drugs, commonly referred as "Biological," it failed on the promise therapeutic of anti-Id vaccines.

Affinity-matured variants derived from nimotuzumab keep the original fine specificity and exhibit superior biological activity.

Nimotuzumab is a humanized monoclonal antibody against the Epidermal Growth Factor Receptor with a long history of therapeutic use, recognizing an epitope different from the ones targeted by other antibodies against the same antigen. It is also distinguished by much less toxicity resulting in a better safety profile, which has been attributed to its lower affinity compared to these other antibodies. Nevertheless, the ideal affinity window for optimizing the balance between anti-tumor activity and toxic effects has not been determined. In the current work, the paratope of the phage-displayed nimotuzumab Fab fragment was evolved in vitro to obtain affinity-matured variants. Soft-randomization of heavy chain variable region CDRs and phage selection resulted in mutated variants with improved binding ability. Two recombinant antibodies were constructed using these variable regions, which kept the original fine epitope specificity and showed moderate affinity increases against the target (3-4-fold). Such differences were translated into a greatly enhanced inhibitory capacity upon ligand-induced receptor phosphorylation on tumor cells. The new antibodies, named K4 and K5, are valuable tools to explore the role of affinity in nimotuzumab biological properties, and could be used for applications requiring a fine-tuning of the balance between binding to tumor cells and healthy tissues.

Antigenic Fingerprinting of Respiratory Syncytial Virus (RSV)-A-Infected Hematopoietic Cell Transplant Recipients Reveals Importance of Mucosal Anti-RSV G Antibodies in Control of RSV Infection in Humans.

BACKGROUND: Respiratory syncytial virus (RSV) infection causes significant morbidity in hematopoietic cell transplant (HCT) recipients. However, antibody responses that correlate with recovery from RSV disease are not fully understood. METHODS: In this study, antibody repertoire in paired serum and nasal wash samples from acutely RSV-A-infected HCT recipients who recovered early (<14 days of RSV shedding) were compared with late-recovered patients (≥14 days of shedding) using gene fragment phage display libraries and surface plasmon resonance. RESULTS: Anti-F serum responses were similar between these 2 groups for antibody repertoires, neutralization titers, anti-F binding antibodies (prefusion and postfusion proteins), antibody avidity, and binding to specific antigenic sites. In contrast, nasal washes from early-recovered individuals demonstrated higher binding to F peptide containing p27. While the serum RSV G antibody repertoires in the 2 groups were similar, the strongest difference between early-recovered and late-recovered patients was observed in the titers of nasal wash antibodies, especially binding to the central conserved domain. Most importantly, a significantly higher antibody affinity to RSV G was observed in nasal washes from early-recovered individuals compared with late-recovered HCT recipients. CONCLUSIONS: These findings highlight the importance of mucosal antibodies in resolution of RSV-A infection in the upper respiratory tract.

Cooperation of intrathymic T15 idiotype-bearing B and complementary T cells in ontogeny of natural Treg cells involved in establishment of T15 clonal dominance.

The mechanisms for dominant T15 idiotype selection are not well understood, yet the significance of idiotypic regulation has been suggested. We proposed that to become dominant V regions of a given subset of B-1a cell must establish a functional idiotypic network with complementary T cells. Features required for the cells involved in immune network and steps preceding the establishment of clonal dominance are suggested.

Monitoring multiple myeloma by idiotype-specific peptide binders of tumor-derived exosomes.

Tumor-derived exosomes (TDEs) play a pivotal role in tumor establishment and progression, and are emerging biomarkers for tumor diagnosis in personalized medicine. To date, there is a lack of efficient technology platforms for exosome isolation and characterization. Multiple myeloma (MM) is an incurable B-cell malignancy due to the rapid development of drug-resistance. MM-released exosomes express the immunoglobulin B-cell receptor (Ig-BCR) of the tumor B-cells, which can be targeted by Idiotype-binding peptides (Id-peptides). In this study, we analyzed the production of MM-released exosomes in the murine 5T33MM multiple myeloma model as biomarkers of tumor growth. To this end, we selected Id-peptides by screening a phage display library using as bait the Ig-BCR expressed by 5T33MM cells. By FACS, the FITC-conjugated Id-peptides detected the MM-released exosomes in the serum of 5T33MM-engrafted mice, levels of which are correlated with tumor progression at an earlier time point compared to serum paraprotein. These results indicate that Id-peptide-based recognition of MM-released exosomes may represent a very sensitive diagnostic approach for clinical evaluation of disease progression.

Description and characterization of a unique human immunoglobulin G1 kappa idiotype found in placental tissue.

Does maternal IgG found in placental tissue provide the fetus with more than just humoral immunity? To address this question, the IgGs from twelve placentas were studied and four of these samples were examined using mass spectrometry which revealed an IgG1k idiotype. A special dodecapeptide portion of the 3rd framework region of the VH chain sequence was identified as an idiotypic determinant in these placental- IgG1k (p-IgG1k) and referred to as peptideX2 and found to have biological activity. Antiserum to peptideX2 was made and then used with Western Immunoblotting to show that this unique H chain (containing peptideX2) appears to be present in all p-IgG tested and in all subjects tested. It appears that the placenta contains not only conventional polyclonal maternal IgGs but also an idiotypic population of maternal IgG1k which binds to TLR2>TLR4 via the epitope "peptideX2″ and promotes IL-6, TNFα, and IL-10 production and may play a role in maternal-fetal tolerance.

Vaccine strategies for the treatment of lymphoma: preclinical progress and clinical trial update.

The clonal B-cell immunoglobulin idiotype found on the surface of lymphomas was the first targeted tumor-specific antigen, and combinations of idiotype with classical and novel adjuvants were shown to stimulate robust humoral and cellular responses, though clinical efficacy was more variable. Cellular and in situ vaccination to help target a wider array of tumor-specific antigens have also been able to stimulate tumor-specific cellular responses, though their clinical success has also been limited. Our growing understanding of the role of regulatory cells and the immunosuppressive tumor microenvironment, along with a wide variety of immunomodulatory agents developed as of late, offer promising adjuvants to potentiate the immune responses elicited by these vaccine protocols and to achieve durable remissions.

Idiotypic DNA vaccination for the treatment of multiple myeloma: safety and immunogenicity in a phase I clinical study.

We report on the safety and immunogenicity of idiotypic DNA vaccination in a phase I, non-randomised, open-label study in patients with multiple myeloma. The study used DNA fusion gene vaccines encoding patient-specific single chain variable fragment, or idiotype (Id), linked to fragment C (FrC) of tetanus toxin. Patients in complete or partial response following high-dose chemotherapy and autologous stem cell transplant were vaccinated intramuscularly with 1 mg DNA on six occasions, beginning at least 6 months post-transplant; follow-up was to week 52. Fourteen patients were enrolled on study and completed vaccinations. Idiotypic DNA vaccines were well tolerated with vaccine-related adverse events limited to low-grade constitutional symptoms. FrC- and Id-specific T-cell responses were detected by ex vivo ELISPOT in 9/14 and 3/14 patients, respectively. A boost of pre-existing anti-FrC antibody (Ab) was detected by ELISA in 8/14 patients, whilst anti-Id Ab was generated in 1/13 patients. Overall, four patients (29 %) made an immune response to FrC and Id, with six patients (43 %) responding to FrC alone. Over the 52-week study period, serum paraprotein was undetectable, decreased or remained stable for ten patients (71 %), whilst ongoing CR/PR was maintained for 11 patients (79 %). The median time to progression was 38.0 months for 13/14 patients. Overall survival was 64 % after a median follow-up of 85.6 months.

Monomeric IgA can be produced in planta as efficient as IgG, yet receives different N-glycans.

The unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. However, in contrast to IgG, reports on plant production of IgA are scarce. We produced IgA1κ and IgG1κ versions of three therapeutic antibodies directed against pro-inflammatory cytokines in Nicotiana benthamiana: Infliximab and Adalimumab, directed against TNF-α, and Ustekinumab, directed against the interleukin-12p40 subunit. We evaluated antibody yield, quality and N-glycosylation. All six antibodies had comparable levels of expression between 3.5 and 9% of total soluble protein content and were shown to have neutralizing activity in a cell-based assay. However, IgA1κ-based Adalimumab and Ustekinumab were poorly secreted compared to their IgG counterparts. Infliximab was poorly secreted regardless of isotype backbone. This corresponded with the observation that both IgA1κ- and IgG1κ-based Infliximab were enriched in oligomannose-type N-glycan structures. For IgG1κ-based Ustekinumab and Adalimumab, the major N-glycan type was the typical plant complex N-glycan, biantennary with terminal N-acetylglucosamine, ß1,2-xylose and core α1,3-fucose. In contrast, the major N-glycan on the IgA-based antibodies was xylosylated, but lacked core α1,3-fucose and one terminal N-acetylglucosamine. This type of N-glycan occurs usually in marginal percentages in plants and was never shown to be the main fraction of a plant-produced recombinant protein. Our data demonstrate that the antibody isotype may have a profound influence on the type of N-glycan an antibody receives.

Translational medicine in action: anti-CD20 therapy in lymphoma.

The introduction of rituximab for B cell lymphoma in the late 1990s inaugurated a new era of cancer therapy showcasing mAbs. mAbs are in principle an amalgamation of two characteristics of a perfect anticancer drug. First, rituximab is a therapy targeted to the tumor cell, but it carries fewer side effects than does chemotherapy. Second, with its ability to directly engage the host immune system, it could potentially elicit longer lasting anticancer immunity, although this remains to be proven. This review highlights the fundamental scientific discoveries that allowed the development of clinically successful anti-CD20 mAbs. Since the approval of rituximab, a considerable amount of work has been undertaken by different groups trying to understand the workings and limitations of anti-CD20s. All of these efforts will be critical in designing new mAbs to CD20 and other targets and, ultimately, of anticancer mAbs that will improve on, or even replace, chemotherapy.

Active idiotypic vaccination versus control immunotherapy for follicular lymphoma.

PURPOSE: Idiotypes (Ids), the unique portions of tumor immunoglobulins, can serve as targets for passive and active immunotherapies for lymphoma. We performed a multicenter, randomized trial comparing a specific vaccine (MyVax), comprising Id chemically coupled to keyhole limpet hemocyanin (KLH) plus granulocyte macrophage colony-stimulating factor (GM-CSF) to a control immunotherapy with KLH plus GM-CSF. PATIENTS AND METHODS: Patients with previously untreated advanced-stage follicular lymphoma (FL) received eight cycles of chemotherapy with cyclophosphamide, vincristine, and prednisone. Those achieving sustained partial or complete remission (n=287 [44%]) were randomly assigned at a ratio of 2:1 to receive one injection per month for 7 months of MyVax or control immunotherapy. Anti-Id antibody responses (humoral immune responses [IRs]) were measured before each immunization. The primary end point was progression-free survival (PFS). Secondary end points included IR and time to subsequent antilymphoma therapy. RESULTS: At a median follow-up of 58 months, no significant difference was observed in either PFS or time to next therapy between the two arms. In the MyVax group (n=195), anti-Id IRs were observed in 41% of patients, with a median PFS of 40 months, significantly exceeding the median PFS observed in patients without such Id-induced IRs and in those receiving control immunotherapy. CONCLUSION: This trial failed to demonstrate clinical benefit of specific immunotherapy. The subset of vaccinated patients mounting specific anti-Id responses had superior outcomes. Whether this reflects a therapeutic benefit or is a marker for more favorable underlying prognosis requires further study.

Naive idiotope-specific B and T cells collaborate efficiently in the absence of dendritic cells.

Anti-idiotope (anti-Id) Abs have a role in therapy against B cell lymphomas, as inhibitors of pathogenic autoantibodies, and as surrogate Ags for immunization. Despite these observations, the mechanism by which Id(+) Ig generates anti-Id Abs is essentially unknown. To address this issue, we generated a double knock-in mouse that expresses V regions of a somatically mutated anti-Id mAb with intermediate affinity (affinity constant [Ka] = 0.77 × 10(7) M(-1)) for the myeloma protein M315. The anti-Id mice have normal peripheral B cell populations, and allelic exclusion is efficient. Anti-Id B cells from BCR knock-in mice, together with Id-specific CD4(+) T cells from previously established TCR-transgenic mice, enabled us to study Id-specific T cell-B cell collaboration by dilution of transferred cells into syngeneic BALB/c recipients. We show that previously unstimulated (naive) Id-specific B and T cells collaborate efficiently in vivo, even at low frequencies and in the presence of low amounts of Id(+) Ig, resulting in germinal center formation, plasma cell development, and secretion of isotype-switched anti-Id Abs. We further demonstrate that Id-specific T cell-B cell collaboration occurs readily in the absence of adjuvant and is not dependent on Id-presentation by dendritic cells. The results underscore the potency of anti-Id B cells in MHC class II-restricted presentation of Id(+) Ig and suggest that Id-specific T cell-B cell collaboration is of physiological relevance.

Idiotype vaccine production using hybridoma technology.

Non-Hodgkin's lymphoma (NHL) is the most common hematological malignancy both in Europe and in the United States. Follicular lymphoma (FL), a tumor comprised of mature B cells, represents one fourth of all NHL and, despite good response rates to standard treatments, tends to frequently relapse to such an extent that it is still considered incurable. Among several alternative therapeutic options actively being pursued, immunotherapy by idiotypic vaccination is in the forefront of clinical experimental medicine. The idiotype vaccine consists of the tumor-specific immunoglobulin conjugated with keyhole limpet hemocyanin (KLH) and administered together with an adjuvant. Over the last 20 years, researchers have proven that this vaccine can induce specific immune responses. Too, those patients with such responses experience a disease-free survival longer than normally achievable, although these latter results require further confirmation in large clinical trials. Traditionally, idiotype vaccines have been produced through hybridoma technology. In this chapter this technology is described.

Cloning variable region genes of clonal lymphoma immunoglobulin for generating patient-specific idiotype DNA vaccine.

Available therapies for lymphoplasmacytic lymphoma (LPL) provide no survival advantage if started before signs or symptoms of end-organ damage develop; hence, current recommendations are to follow a program of observation while patients are in the asymptomatic phase of disease. We hypothesize that using idiotypic determinants of a B-cell lymphoma's surface immunoglobulin as a tumor-specific marker, we can develop patient-specific chemokine-idiotype fusion DNA vaccines that induce an immune response against LPL. By activating the host immune system against the tumor antigen, we postulate that disease control of asymptomatic phase lymphoplasmacytic lymphoma can be maintained. These chemokine-idiotype fusion DNA vaccines provide protection in a lymphoma mouse model and have recently entered clinical trials. Herein, we describe procedures for the generation of therapeutic vaccines, particularly "second-generation" recombinant vaccines. Specifically, in the Methods section we describe how to identify lymphoma-associated immunoglobulin V (IgV) genes from patient biopsy and how to assemble these genes as single-chain variable gene fragment (scFv) in-frame with MIP-3α to generate novel DNA fusion vaccines.

Cloning of variable fragments of tumor immunoglobulin, assembling and expressing of human SCFV protein in E. coli for anti-idiotype vaccination.

AIM: Idiotype, the unique part of immunoglobulin molecule expressed on the surface of B-cells, represents a specific antigen for vaccination against lymphoma. We have developed a rapid method for immunoglobulin variable fragments cloning, assembling and expression of recombinant idiotype protein in Escherichia coli. METHODS: PCR with specially designed panel of primers was used for direct amplification of variable regions of tumor immunoglobulin. Overlapping extension PCR, restriction and ligation was applied for assembling and cloning of vaccine construction. Idiotype protein was purified by metal-chelate chromatography. RESULTS: Methods of idiotype cloning from lymphoma cells and production of recombinant protein were developed and optimized. Several samples of idiotypic proteins originating from B-cell lines and lymphoma patients were produced. CONCLUSION: The proposed method of vaccine production is relatively cheap, not very laborious and requires as long as 6-7 week to perform. The expressed protein was soluble, did not accumulate in inclusion bodies and harvested at sufficient for vaccination quantity and concentration.