VH IG PEPTIDE VACCINES FOR HUMAN B CELL MALIGNANCIES

人类B细胞恶性肿瘤表面可见膜相关免疫球蛋白(Ig)。免疫球蛋白重链和轻链氨基末端的可变区(VH和VL)含有代表肿瘤特异性抗原的克隆性特异性表位。从B细胞淋巴瘤患者的肿瘤标本中克隆和测序VH cDNA使我们能够根据这些序列产生不同的合成肽。这将被用来测试疫苗接种策略，在这种策略中，患者的树突状细胞被这些多肽冲击，并将检测它们诱导肿瘤特异性免疫的能力。在目标1中，正在测试与具有生殖系或体细胞突变序列的B细胞淋巴瘤的高变区(CDR1-3)相对应的多肽。在AIM中，两个对应于更保守的生殖系和体细胞突变框架VH区域的多肽正在进行免疫原性测试。在前两个疫苗接种策略(目标L和2)中，我们将使用11-30个氨基酸的合成肽来冲击DC，这些DC有望(通过巨噬细胞吞噬)在MHC Class 1和11的背景下被摄取、加工和呈递给自体淋巴细胞。在目标3中，从整个肿瘤相关的VH区域中选择较小的合成肽(9-MERS)，它们预计不经过处理就直接与树突状细胞上的I类结合，这些结合强度是由患者表达的HLA等位基因的多肽结合基序预测的。在目标4中，将编码选定的VH区肽或整个肿瘤相关VH区的哺乳动物表达载体导入患者的树突状细胞，并对转基因的树突状细胞进行免疫原性测试。在所有4个AIMS中，使用人/SCID小鼠嵌合模型评估了疫苗策略诱导保护性抗肿瘤免疫的能力，在该模型中，患者的多肽脉冲或转基因的树突状细胞与患者的淋巴细胞一起植入SCID小鼠，然后用自体肿瘤细胞攻击。多肽冲击或转染树突状细胞也在体外评估它们激发肿瘤特异性细胞毒性T淋巴细胞的能力。这些结果有望确定免疫原性VH区肽，并为B细胞恶性肿瘤患者的疫苗接种建立最佳的临床策略。在为已确定肿瘤特异性抗原的其他肿瘤设计疫苗接种方案方面，也有望获得有价值的见解。

Human B-cell malignancies display on their surface, membrane-associated immunoglobulin (Ig). The variable regions at the amino terminal ends of both heavy and light chains (VH and VL) of Ig contain clonal specific epitopes that represent tumor specific antigens. The cloning and sequencing of VH cDNAs from tumor specimens of patients with B-cell lymphomas allow us to generate different synthetic peptides based upon these sequences. This will be exploited to test vaccination strategies in which patients' dendritic cells are pulsed with these peptides and their ability to induce tumor specific immunity will be examined. In Aim 1, peptides corresponding to hypervariable (CDR1-3) regions of B-cell lymphomas that have either germline or somatically mutated sequences are being tested. In Aim 2 peptides corresponding to the more conserved germline and somatically mutated framework VH regions are being tested for immunogenicity. In these first two vaccination strategies (Aims l and 2) we will use synthetic peptides of 11-30 amino acids to pulse DC's which would be expected to take up (by macropinocytosis), process and present small peptides in the context of MHC Class 1 and ll to autologous lymphocytes. In Aim 3, smaller synthetic peptides (9-mers) which are expected to bind directly to Class I on dendritic cells without processing, are selected from the entire tumor-associated VH region based upon optimal MHC Class I binding strengths that are predicted by peptide binding motifs specific for an HLA allele expressed by the patient. In Aim 4, patients' dendritic cells are transfected with mammalian expression vectors encoding either selected VH region peptides or the entire tumor associated VH region and the transfected dendritic cells tested for immunogenicity. In all 4 Aims, the vaccination strategies are evaluated for their ability to induce protective anti-tumor immunity using a human/SCID mouse chimeric model in which patients' peptide-pulsed or transfected dendritic cells, along with patients' lymphocytes, are engrafted in SCID mice and subsequently challenged with autologous tumor cells. Peptide pulsed or transfected D.C. are also evaluated in vitro for their ability to provoke tumor specific cytotoxic T lymphocytes. These results are expected to define immunogenic VH region peptides and to establish an optimal clinical strategy for the vaccination of patients with B cell malignancies. Valuable insights are also anticipated with respect to designing vaccination protocols for other tumors where a tumor specific antigen has been identified.