IDIOTOPE-SPECIFIC LAMPREY ANTIBODY. using a 5 primer containing a XmaI site, CLL655_VH_FR1_XmaI_Fwd

Transcription

1 NAKAHARA et al IDIOTOPE-SPECIFIC LAMPREY ANTIBODY Supplementary Materials and Methods Cloning of CLL donor BCR as scfv. Briefly, for the first round of PCR, the VH region was amplified using a primer containing a XmaI site, CLL6_VH_FR_XmaI_Fwd (GTCCTCGCAACTGCCCCATCCCGGGGCCCAACCAGCGATGGCCCAGGTGCAGCTGGTGCAG TCTGG) and primer containing a (Gly -Ser) linker sequence, CLL6_VH_scFv_Rev (CCGCCGGATCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTGAC) with the following cycle profile: 9 C for min, (9 C, min; C, min; and 7 C, min), 7 C for 7 min. For VL region amplification, a primer containing a (Gly -Ser) linker sequence, CLL6_VL_scFv_Fwd (GGAGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCGGACATCCAGATGACCCAG TCTCC) and primer containing a SalI site and FLAG tag, CLL6_VL_FR_SalI_Rev (GAGTCATTCTCGACTGCTATGTCGACTTTATCATCATCATCTTTATAATCACGTTTGATATCC ACTTTGGT) were used with the following cycle profile: 9 C for min, (9 C, min; C, min; and 7 C, min), 7 C for 7 min. For the second round of PCR, the scfv gene was assembled by overlap-extension based on the (Gly -Ser) linker sequence homology between the VH and VL PCR products using the following cycle profile: 9 C for min, (9 C, min; C, min; and 7 C, min) 6, 7 C for 7 min. In the third round of PCR, the assembled scfv gene was PCR amplified using the outside primers, CLL6_VH_FR_XmaI_Fwd and CLL6_VL_FR_SalI_Rev using the following cycle profile: 9 C for min, (9 C, min; C, min; and 7 C, min), 7 C for 7 min. PCR products were purified by QIAquick Gel Extraction Kit (Qiagen), digested with XmaI and SalI (New England Biolabs), and cloned into pdisplay vector (Invitrogen) using T DNA ligase (New England Biolabs). CLL donor scfv plasmid was purified from transformed NovaBlue competent cells (Novagen) with the QIAprep Spin Miniprep Kit (Qiagen).

2 NAKAHARA et al IDIOTOPE-SPECIFIC LAMPREY ANTIBODY Construction of scfv mutants. The two scfv mutants were constructed by amplifying the VH and VL regions from the wildtype CLL donor scfv plasmid utilizing overlap-extension PCR as described above, except using different primer sets for the first round of PCR. For the HCDR Swap scfv mutant, the VH region was amplified using a primer containing a XmaI site, CLL6_VH_FR_XmaI_Fwd (GTCCTCGCAACTGCCCCATCCCGGGGCCCAACCAGCGATGGCCCAGGTGCAGCTGGTGCAG TCTGG) and primer containing the CLL VH CDR sequence as overhang, CLL6_FR- _CDR_Rev (TTGTTGATAATAACTCCCTGAACCATAGGACGCCTCTCTCGCACAGTAATA ). The VL region was amplified using a primer containing the CLL VH CDR sequence as overhang, CLL_CDR-6_FR_Fwd (GAGGCGTCCTATGGTTCAGGGAGTTATTATCAACAATACTTTGACTACTGG) and primer containing a SalI site and FLAG tag, CLL6_VL_FR_SalI_Rev (GAGTCATTCTCGACTGCTATGTCGACTTTATCATCATCATCTTTATAATCACGTTTGATATCC ACTTTGGT). The HCDR Swap scfv gene was assembled by overlap-extension based on the CLL VH CDR sequence homology between the VH and VL PCR products. For the Donor HCDR Swap scfv mutant, the VH region was amplified using a primer containing a XmaI site, CLL6_VH_FR_XmaI_Fwd (GTCCTCGCAACTGCCCCATCCCGGGGCCCAACCAGCGATGGCCCAGGTGCAGCTGGTGCAG TCTGG) and primer containing the CLL VH FR sequence as overhang, CLL6_CDR- _FR_Rev (TGAGGAGACGGTGACCGTGGTCCCTTTGCCCCAGACGTCCATGTAGTAGTAGTAGTAGTAG TACCCCCAAAAATC). The VL region was amplified using a primer containing the CLL VH FR sequence as overhang and end of a (Gly -Ser) linker sequence, CLL_FR-GlySer_Fwd (TACTACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCAG GTGGAGGCGGTTCA) and primer containing a SalI site and FLAG tag, CLL6_VL_FR_SalI_Rev

3 NAKAHARA et al IDIOTOPE-SPECIFIC LAMPREY ANTIBODY (GAGTCATTCTCGACTGCTATGTCGACTTTATCATCATCATCTTTATAATCACGTTTGATATCC ACTTTGGT). The Donor HCDR Swap scfv gene was assembled by overlap-extension based on the (Gly -Ser) linker sequence homology between the VH and VL PCR products Quantitative real-time PCR. RNA from CLL donor MNCs were converted into cdna as described in Materials and Methods. Quantitative real-time PCR was done using the Customized TaqMan Gene Expression Assay (Invitrogen) on a LightCycler Instrument II (Roche). The sequences for the upstream IGHV allele-specific primer, downstream IGHJ allele-specific primer, and the TaqMan probe complementary to the hypervariable V N D region of the CLL donor HCDR are as follows: upstream primer (GCTGAGCAGCCTGAGATCTG), downstream primer (GGCCCCAGTAGTCAAAGTAGTAC), and probe (CTGTGCGAGAGTTACAGTCAAG). Two independent quantitative real-time PCR reactions with duplicate samples were performed, and the value of the target gene was normalized to the glyceraldehyde -phosphate dehydrogenase (GAPDH) gene. The fold change in the CLL donor HCDR transcript relative to the GAPDH endogenous control was determined by the following formulas: Fold Change = - T = C T, CLL donor HCDR -C T, GAPDH 6 T T, stimulated - T, control

4 NAKAHARA et al IDIOTOPE-SPECIFIC LAMPREY ANTIBODY Supplementary Figure Legends Figure S: Flow cytometric analysis of monoclonal VLR9 reactivity with different cell types and cell lines. VLR9 binding (red line) was compared with that of control VLR having Bacillus anthracis BclA-specificity (gray shading). Histogram of VLR9 binding to (A) five different healthy donor lymphocytes gated on B cells (CD - /CD9 + ), T cells (CD + /CD9 - ), and non-b/t cells (CD - /CD9 - ), (B) four EBV transformed B cells, and (C) four different human B cell leukemia/lymphoma lines, including pre-b cell leukemia (697), Burkitt s Lymphoma (Daudi, Ramos), and diffuse large B cell lymphoma (SU- DHL-6). 9 Table S: CLL VH gene families and HCDR sequences. The IGHV, IGHD, and IGHJ genes and the HCDR amino acid sequences expressed in the leukemic cells of 6 CLL patients tested for VLR9 reactivity are shown. Shared VH gene family usage between CLL donor and other CLL patients are highlighted in yellow (IGHV), blue (IGHD), and green (IGHJ). 6 Figure S: Flow cytometric analysis of monoclonal VLR9 reactivity with CLL cells with different VH gene sets. VLR9 binding (red line) was compared with that of control VLR having Bacillus anthracis BclA-specificity (gray shading). Histogram of VLR9 binding to 6 CLL patients with known VH gene sequences (gated on CD + /CD9 + lymphocytes) Figure S: CLL VH gene sequence analysis. Sequence alignment between the IGVH-D-J gene rearrangements of CLL donor and those of the 6 CLL patients tested for VLR9 reactivity by flow cytometry are shown. Shared VH gene family usage between CLL donor and other CLL patients are highlighted in yellow (IGVH), blue (IGHD), and green (IGHJ). Identical CDR, CDR, and IGHJ amino acid sequences are shown in red. Figure S: IGHV-D-J sequence alignment of VLR9 sorted cells. Sequence alignment between the IGHV-D-J gene rearrangement of CLL donor and those of the VLR9 + /CD hi cells sorted from the CLL

5 NAKAHARA et al IDIOTOPE-SPECIFIC LAMPREY ANTIBODY donor B cells at 8 months after treatment. No significant alignment was possible for the VLR9 - /CD lo cells. Nucleotide identity to the original CLL donor IGHV-D-J sequencing results is highlighted in gray. 6 7 Table S: Detection of CLL recurrence by quantitative real-time PCR. Cryopreserved MNCs from the CLL donor after treatment were analyzed by qpcr for the presence of CLL donor HCDR transcript. Each time point is calculated as months after end of CFAR treatment regimen. The C T T T ) of the qpcr reaction using the CLL donor HCDR-specific probe and GAPDH probes were used to calculate the fold change in the CLL donor HCDR transcript relative to the GAPDH endogenous control.

6 Figure S A B Cell T Cell Non-B/T Cell YD-H YD-A Healthy Donors: YD YD YD6 % of Max VLRB B EBV Transformed B Cells: WT WT6 M7 M7 % of Max VLRB C Human B Cell Lines: 697 Daudi Ramos SU-DHL-6 % of Max VLRB

7 Table S. CLL VH gene families and HCDR sequences CLL ID IGHV Gene IGHD Gene IGHJ Gene HCDR amino acid sequence Donor IGHV-69* IGHD-* IGHJ* ARVTVKYYDFWGYYFDY IGHV-69* IGHD-* IGHJ* DDSYYDFWSGWYY IGHV-69* IGHD-* IGHJ6* ARVEIFGVVGLSYYYYYMDV 6 IGHV-69* IGHD-* IGHJ6* ARGAIFGVVIIPVTPFYMDV 89 IGHV-9* IGHD-* IGHJ* AKDASSNYDFWSGYYDY 7 IGHV-b* IGHD-* IGHJ* ARVMEKYYDFWSGYYYFD IGHV-69* IGHD-* IGHJ6* AREASYGSGSYYQQYYYYYYMDV IGHV-69* IGHD-* IGHJ6* AVGVLWFGELLFSYYYYYGMD IGHV-69* IGHD-* IGHJ6* AGRLIFGVVITAGGDYGMDV IGHV-* IGHD-* IGHJ* ARAPIGSTIFGVVIIRFAFDI 76 IGHV-* IGHD-* IGHJ* ARTQIGDCGGDCYPFDY 6 IGHV-* IGHD6-9* IGHJ* AREQWLVLSYFDY 6 IGHV-* IGHD-* IGHJ* ARDLRYSYGSGSTPFLDS 78 IGHV-8* IGHD-6* IGHJ* ARKSWVGAYYFDY 8 IGHV-* IGHD-* IGHJ* VSHYCTSSTCDQMY IGHV-* IGHD6-9* IGHJ* AREQWLASPNLDY 7 IGHV-* IGHD6-6* IGHJ* ARGRWSPKFVL 99 IGHV-*/IGHV-* IGHD-* IGHJ* AKGLVIGLPDV IGHV-7* IGHD-8* IGHJ* ARSSRDGTNDYDGEYRYFDY IGHV-*/IGHV-*6 IGHD-9* IGHJ* ATRPQLNYDILTGYYIGGGYFDL 9 IGHV-* IGHD7-7* IGHJ6* ARDPYRGLYGMFYFYYMDV 7 IGHV-9* IGHD-* IGHJ* ARNPYCGGDCYSDAFDI 9 IGHV-* IGHD-* IGHJ6* ARDLYGWTYCSSTSCYRYYGMDV 6 IGHV-* IGHD-* IGHJ6* ARDRVDIVATTTYYYYYYGMDV IGHV-69* IGHD-* IGHJ* ARSTNLDYFFAAVTGNWFDP 8 IGHV-* IGHD-8* IGHJ6* TRAMDYYYGMDV 97 IGHV-69*6 IGHD-* IGHJ6* ATPPRGTYDSSGYYYGGLDNYYGMDV

8 Figure S CLL Patients: % of Max VLRB

9 Figure S < FR ><--CDR--><------FR------><-CDR->< FR FR------>< CDR ><---FR---> Donor QVQLVQSGAEVKKPGSSVKVSCKASGGTFSS..YAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARVT...VKYYDFWGYYFDYWGQGTLVTVSS QVQLVQSGAEVKKPGSSVKVSCKASGGTFSS..YAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDDSYYD...FW..SGWYYWGQGTLVTVSS QVQLVQSGAEVKKPGSSVKVSCKASGGTFSS..YAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARVEIFGV...VGLSYYYYYMDVWGKGTTVTVSS 6 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSS..YAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGAIFGV...VIIPVTPFYMDVWGKGT EVQLVESGGGLVQPGRSLRLSCAASGFTFDD..YAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDASSNYD...FWSGYYDYWGQGTLVTVSS 7 QVQLQESGPGLVKPSETLSLTCTVSGYSISSG.YYWGWIRQPPGKGLEWIGSIYHSGS.TYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVMEKY...YDFWSGYYYFDYWGQGT... QVQLVQSGAEVKKPGSSVKVSCKASGGTFSS..YAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREASYGSG...SYYQQYYYYYYMDVWGKGT... QVQLVQSGAEVKKPGSSVKVSCKASGGTFSS..YAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAVGVLWFG...ELLFSYYYYYGMDVWGQGT... QVQLVQSGAEVKKPGSSVKVSCKASGGTFSS..YAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAGRLIFGV...VITAGGDYGMDVWGQGTTVTVSS QVQLQESGPGLVKPSQTLSLTCAVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGS.TYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAPIGST..I...FGVVIIRFAFDIWGQGTMVTVSS 76 QVQLVQSGAEVKKPGASVKVSCKASGYTFAA..YYMHWVRQAPGQGLEWMGWINPNTGGTTYAQKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYSCARTQIGDCGG...DCYPFDYWGQGTLVTVSS 6 QVQLVQSGAEVKNPGASVKVSCKASGYTFIT..YAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREQWLVLS...YFDYWGQGTLVTVSS 6 QVHLTQSGAEVKKPGASVKVSCKASGYTFSG..YYLHWVRQAPGQGLEWMGWINPNGGGTNYAQKFQGWVTLTRDTSISTAYMEMRRLTSDDTAVYFCARDLRYSYGSG...STPFLDSWGQGTLVTVTS 78 QVQLVQSGAEVKKPGASVKVSCKASGYTFTS..YGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARKSWVGAY...YFDYWGQGTLVTVSS 8 QVRLVQSGAEVKKPGASVKASCKTSGYTFTA..YAVHWVRQAPGQRLECMGWVNPANADTGYSQKFQGRLTITRDISASTVHMELSSLRSEDTAVYYCVSHYCTSSTCD...QMYWGQGTLVTVSS QVQLVQSGAEVKKPGASVKVSCKASGYTFTG..YYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREQWLASP...NLDYWGQGTLVTVSS 7 QVQLQQWGAGLLKPSETLSLSCAVYGGSLSG..FYWSWIRQPPGKGLEWIGDINPSGG.TNYNPSLKSRVTILADTSRNQFSLKVSSVTAADTAVYYCARGRWSP...KFVLWGQGTLVTVSS 99 EVQLLESGGDLVQPGGSLRLSCAASGFTFSS..YAMTWVRQAPGKGLEWVSAISGSGGSTNYGDSVKGRFTISRDNSNNTLYLQMDSLRPEDTAVYYCAKG...LVIGLPDVWGQGTSVTVSS EMQLVESGGGLVQPGGSLRLSCAASGFTFSR..YWMTWVRQAPGKGLEWVANIKQDGSERYYVGSVKGRFTISRDNAKNSLSLQMNSLRADDTAVYYCARS...SRDGT...NDYDGEYRYFDYWGQGTTVTVSS QVQLVESGGGVVQPGRSLRLSCAASGFTFSS..YGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATRPQLNYDIL...TGYYIGGGYFDLWGRG... 9 EVQLVESGGGLVMPGGSLRLSCAASGFIFSD..YNMNWVRLAPGSGLEWVSSITTNSGHIYYADSVKGRFTISRDNAQNSLYLQMNILTAEDTAVYFCARDPYRGLYGM...FYFYYMDVWGKGTTVTVSS 7 QVQLQESGPGLVKPSETLSLTCTVSGGSISS..YYWSWIRQPPGKGLEWIGYIYYSGS.TNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARNPY...CGGDCYSDAFDIWGQGTMVTVS. 9 QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGS.TYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDLYGWTYCS...STSCYRYYGMDVWGQGT... 6 EVQLVESGGGLVQPGGSLRLSCAASGFTVSS..NYMSWVRQAPGKGLEWVSVIYSGGS.TYYADSVKGRFTISRHNSKNTLYLQMNSLRAEDTAVYYCARDR.VDIVAT...TTYYYYYYGMDVWGQGT... QVQLVQSGAEVKKPGSSVKVSCKASGGTFSS..YTISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSTN.LD...YFFAAVTGNWFDPWGQGT... 8 QVLLQQWGAGLLEPSDTLSLTCAVYGGSLSG..YYWIWIRQSPGKGLEWIGKIKYSGS.TDYNPSLKSRVTISIDTSKKQFSLKLSCVTAADTAVYYCTRAMD...YYYGMDVWGPGTMVTVSS 97 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSS..YAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCATPPRGTYDSSGYYYGGLDNYYGMDVWGQGTTVTVSS

10 Figure S < FR >< CDR >< Donor CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTA VLR9 +...TGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTA FR >< CDR >< FR Donor TCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAG VLR9 + TCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAG FR ><---CDR--- Donor AGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGTTACA VLR9 + AGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGTTACA CDR >< FR > 7 Donor GTCAAGTATTACGATTTTTGGGGGTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA VLR9 + GTCAAGTATTACGATTTTTGGGGGTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC...

11 Table S. Detection of CLL recurrence by quantitative real-time PCR Months After End of CFAR Treatment Donor CLL HCDR (C T ) GAPDH (C T ) T T ) Fold Change CLL HCDR Detection 8 not detectable 8.7 ±.. negative 7.8. ±.... weak positive 6 6. ±. 8. ± positive