VH IG PEPTIDE VACCINES FOR HUMAN B CELL MALIGNANCIES

人B细胞恶性肿瘤在其表面上显示膜相关免疫球蛋白（IG）。在IG的重链和轻链（VH和VL）的氨基末端的可变区含有代表肿瘤特异性抗原的克隆特异性表位。来自B细胞淋巴瘤患者的肿瘤标本的VH cDNA的克隆和测序允许我们基于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，其中患者的树突状细胞用这些肽脉冲，并将检查它们诱导肿瘤特异性免疫的能力。在目标1中，测试了对应于具有种系或体细胞突变序列的B细胞淋巴瘤的高变（CDR 1 -3）区的肽。在目标2中，正在测试对应于更保守的种系和体细胞突变的框架VH区的肽的免疫原性。在这前两种疫苗接种策略（目的1和2）中，我们将使用11-30个氨基酸的合成肽来脉冲DC，预期DC在MHC 1类和II类的背景下摄取（通过巨胞饮作用）、加工和呈递小肽至自体淋巴细胞。在目的3中，基于通过对患者表达的HLA等位基因特异性的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区中选择预期直接结合树突细胞上的I类而不经加工的较小合成肽（9聚体）。在目的4中，用编码所选VH区肽或整个肿瘤相关VH区的哺乳动物表达载体转染患者的树突细胞，并测试转染的树突细胞的免疫原性。在所有4个目的中，使用人/SCID小鼠嵌合模型评价疫苗接种策略诱导保护性抗肿瘤免疫的能力，其中将患者的肽脉冲或转染的树突状细胞与患者的淋巴细胞一起沿着移植到SCID小鼠中，随后用自体肿瘤细胞攻击。还在体外评价肽脉冲或转染的D.C.激发肿瘤特异性细胞毒性T淋巴细胞的能力。预期这些结果将确定免疫原性VH区肽，并建立B细胞恶性肿瘤患者疫苗接种的最佳临床策略。对于已经确定肿瘤特异性抗原的其他肿瘤，也可以预期在设计疫苗接种方案方面有价值的见解。

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.