VH Peptide Vaccine Strategies for B Cell Lymphomas

免疫球蛋白（1g）可变区是B细胞淋巴瘤和骨髓瘤的肿瘤相关抗原。用完整的IG疫苗接种诱导了肿瘤特异性免疫，但这种方法产生了不同的结果，并导致了肿瘤变体的生长。我们推测，所见的可变结果可能是由于当使用全IG作为疫苗时，对单一或非常有限的免疫显性表位集合引发有限的免疫应答。因此，我们的目标是定义代表来自超变和保守框架区的亚显性或隐蔽表位的离散VH肽，以设计可以引起更广泛应答的疫苗接种策略。此外，可以将激发针对包括VH框架区在内的更保守区域内的表位的保护性免疫的疫苗用于多个患者。我们的策略是从整个VH区鉴定具有预测的H-2Kd结合亲和力的肽，并测试它们产生MHC限制性CTL的能力。用用合成肽脉冲的树突状细胞、用转染以表达所选肽的树突状细胞或用整个VH区转染的树突状细胞免疫小鼠。我们的理由是，通过使用树突状细胞并集中于选定的肽，将有可能产生针对VH区域的CTL应答，否则这些区域将由于更占优势的表位的抑制作用而被忽略。为了验证我们的假设，将BALB/c小鼠对葡聚糖和邻苯二甲酸半抗原的免疫应答产生的抗体形成克隆永生化为杂交瘤，并将相关的VH区肽用于评估T细胞库和用作肿瘤模型的杂交瘤细胞的特异性。这两种模型的优势在于：（a）每个杂交瘤代表初级或次级应答中的显性克隆型，和（B）这些杂交瘤的Vh区已被测序，并已鉴定出来自这些区域的MHC结合肽。虽然我们期望看到肿瘤特异性MHC限制性CTL对生殖系和体细胞突变肽的应答，但正在尝试通过将肽与热休克蛋白连接，通过从生物可降解微球局部释放细胞因子以及通过用含CpG的寡脱氧核苷酸处理树突状细胞来增强应答。疫苗接种的功效在体外评估，并且还通过评估免疫小鼠耐受肿瘤攻击或预防缓解期肿瘤生长的能力在体内测试。最后，评估疫苗接种对正常B细胞库表达的影响。

Immunoglobulin (1g) variable regions are tumor associated antigens of B cell lymphomas and meyelomas. Vaccination with intact Ig has induced tumor specified immunity, but this approach has produced variable results and has led to the outgrowth of tumor variants. We hypothesize that the variable results seen may be due to the elicitation of a limited immune response to single or very restricted set of immunodominant epitopes when whole Ig is used as the vaccine. Our goal is therefor to define discrete VH peptides representing subdominant or cryptic epitopes from both the hyper variable and conserved framework regions to design a vaccination strategy that could provoke a broader response. In addition a vaccine that provokes protective immunity against epitopes within more conserved regions including framework regions of VH could be utilized for multiple patients. Our strategy is to identify peptides from he entire VH region with predicted binding affinities for H-2Kd, and test them for their ability to generate MHC-restricted CTL. Mice are immunized with dendritic cells pulsed with synthetic peptides, with dendritic cells transfected to express the selected peptides, or with dendritic cells transfected with the entire VH region. Our rational is that by using dendritic cells and focusing upon selected peptides it will be possible to generate CTL responses to areas of the VH that would otherwise be ignored due to the suppressive effects of more dominate epitopes. To test our hypothesis, antibody forming clones derived from the immune response of BALB/c mice to dextran and to the hapten phthalate have been immortalized as hybridomas and associated VH region peptides will be used to assess the specificity of the T cell repertoire and the hybridoma cells used as tumor models. The strengths of these two models are that (a) each hybidoma represents a dominant clonotype in the primary or secondary response, and (b) the Vh regions of these hybidomas have been sequenced and MHC binding peptides from these regions have been identified. While we expect to see tumor specific MHC-restricted CTL responses to both germ line and somatically mutated peptides an attempt being made to enhance the responses by linking peptides to heat shock proteins, by the local release of cytokines from biodegradable micro spheres and by treatment of dendritic cells with CpG-containing oligodeoxynucleotides. The efficacy of vaccination is evaluated in vitro and also tested in vivo by assessing the ability of immunized mice to withstand a tumor challenge or to prevent the outgrowth of tumor in remission. Finally, the effect of vaccination upon the normal B-cell repertoire expression is assessed.