VH IG PEPTIDE VACCINES FOR HUMAN B CELL MALIGNANCIES

人b细胞恶性肿瘤表面显示膜相关免疫球蛋白（Ig）。Ig重链和轻链（VH和VL）氨基末端的可变区含有克隆特异性表位，代表肿瘤特异性抗原。b细胞淋巴瘤患者肿瘤标本中VH cdna的克隆和测序使我们能够基于这些序列生成不同的合成肽。这将被用于测试疫苗接种策略，其中患者的树突状细胞被这些肽脉冲，它们诱导肿瘤特异性免疫的能力将被检查。在Aim 1中，正在测试具有种系或体细胞突变序列的b细胞淋巴瘤的高变（CDR1-3）区域对应的肽。在Aim 2中，与更保守的种系和体细胞突变框架VH区域相对应的肽正在进行免疫原性测试。在前两种疫苗接种策略（目标1和目标2）中，我们将使用11-30个氨基酸的合成肽来脉冲DC，这些DC将在MHC 1类和ll类的背景下（通过巨噬细胞作用）吸收、处理和呈递小肽到自体淋巴细胞。在Aim 3中，根据患者表达的HLA等位基因特异性肽结合基序预测的最佳MHC I类结合强度，从整个肿瘤相关VH区域中选择较小的合成肽（9-mers），这些肽有望直接与树突状细胞上的I类结合而无需加工。在Aim 4中，用编码选定VH区域肽或整个肿瘤相关VH区域的哺乳动物表达载体转染患者的树突状细胞，并检测转染后的树突状细胞的免疫原性。在所有4个目标中，使用人/SCID小鼠嵌合模型评估了疫苗接种策略诱导保护性抗肿瘤免疫的能力，在该模型中，将患者的肽脉冲或转染的树突状细胞以及患者的淋巴细胞移植到SCID小鼠中，随后用自体肿瘤细胞攻击。肽脉冲或转染dc也在体外评估其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。对于已经鉴定出肿瘤特异性抗原的其他肿瘤的疫苗接种方案的设计，也期望有价值的见解。

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.