VH CDR3 Research Articles

Novel Structural Parameters of Ig-Ag Complexes Yield a Quantitative Description of Interaction Specificity and Binding Affinity.

Antibody–antigen complexes challenge our understanding, as analyses to date failed to unveil the key determinants of binding affinity and interaction specificity. We partially fill this gap based on novel quantitative analyses using two standardized databases, the IMGT/3Dstructure-DB and the structure affinity benchmark. First, we introduce a statistical analysis of interfaces which enables the classification of ligand types (protein, peptide, and chemical; cross-validated classification error of 9.6%) and yield binding affinity predictions of unprecedented accuracy (median absolute error of 0.878 kcal/mol). Second, we exploit the contributions made by CDRs in terms of position at the interface and atomic packing properties to show that in general, VH CDR3 and VL CDR3 make dominant contributions to the binding affinity, a fact also shown to be consistent with the enthalpy–entropy compensation associated with preconfiguration of CDR3. Our work suggests that the affinity prediction problem could be partially solved from databases of high resolution crystal structures of complexes with known affinity.

Reappraising Immunoglobulin Repertoire Restrictions in Chronic Lymphocytic Leukemia: Focus on Major Stereotyped Subsets and Closely Related Satellites

Reappraising Immunoglobulin Repertoire Restrictions in Chronic Lymphocytic Leukemia: Focus on Major Stereotyped Subsets and Closely Related Satellites

Isolation and characterization of a novel human scFv inhibiting EGFR vIII expressing cancers

EGFRvIII, a mutant form of epidermal growth factor receptor is highly expressed in glioblastoma, carcinoma of the breast, ovary, and lung but not in normal cells. This tumor specific antigen has emerged as a promising candidate for antibody based therapy of several cancers. The aim of the present study was isolation and characterization of a human single chain antibody against EGFRvIII as a promising target for cancer therapy. For this, a synthetic peptide corresponding to EGFRvIII protein was used for screening the naive human scFv phage library. Selection was performed using a novel screening strategy for enrichment of rare specific clones. After five rounds of screening, six positive scFv clones against EGFRvIII were selected using monoclonal phage ELISA, among them, a clone with an amber mutation in VH CDR2 coding sequence showed higher reactivity. The mutation was corrected through site directed mutagenesis and then scFv fragment was expressed after subcloning into the bacterial expression vector. Expression in BL21 pLysS resulted in a highly soluble scFv appeared in soluble fraction of E. coli lysate. Bioinformatic in silico analysis between scFv and EGFRvIII sequences confirmed specific binding of desired scFv to EGFRvIII in CDR regions. The specific reactivity of the purified scFv with native EGFRvIII was confirmed by cell based ELISA and western blot. In conclusion, human anti- EGFRvIII scFv isolated from a scFv phage library displayed high reactivity with EGFRvIII. The scFv isolated in this study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFRvIII expressing cancers.

AC-1001 H3 CDR peptide induces apoptosis and signs of autophagy in vitro and exhibits antimetastatic activity in a syngeneic melanoma model.

Antibody‐derived peptides modulate functions of the immune system and are a source of anti‐infective and antitumor substances. Recent studies have shown that they comprise amino acid sequences of immunoglobulin complementarity‐determining regions, but also fragments of constant regions. VH CDR3 of murine mAb AC‐1001 displays antimetastatic activities using B16F10‐Nex2 murine melanoma cells in a syngeneic model. The peptide was cytotoxic in vitro in murine and human melanoma cells inducing reactive oxygen species (ROS) and apoptosis by the intrinsic pathway. Signs of autophagy were also suggested by the increased expression of LC3/LC3II and Beclin 1 and by ultrastructural evidence. AC‐1001 H3 bound to both G‐ and F‐actin and inhibited tumor cell migration. These results are important evidence of the antitumor activity of Ig CDR‐derived peptides.

Chystallographic Evidence of Autologous Recognition By a Clonotypic B Cell Receptor in Chronic Lymphocytic Leukemia

Abstract Chronic lymphocytic leukemia (CLL) leukemic cells express B-cell receptor immunoglobulin (BcR IG) whose signaling is of paramount importance throughout the natural history of the disease. Indeed, signaling pathways downstream of the BcR are constitutively active in all cases of CLL and inhibitors of the Bruton's tyrosine kinase BTK (Ibrutinib) or PI3Kδ (Idelalisib), two downstream signaling effectors, are clinically effective. This functional evidence complements earlier molecular observations supporting antigen drive in CLL ontogeny, including the distinction of CLL into cases with somatically hypermutated BcR IG (M-CLL) that have a significantly better outcome compared to those with unmutated, germline-like receptors (U-CLL). CLL also displays a remarkably skewed BcR IG gene repertoire, culminating in the existence of highly homologous, stereotyped BcR IG in >30% of cases, indicating selection by a limited set of antigenis. A number of potential antigenic elements have been described, being recognized by the monoclonal receptors and able to deliver intracellular signals. More recently, it has been reported that CLL cells are endowed with the apparently unique property of autonomous signaling, since individual CLL-derived BcR IG can promote Ca2+ influx and NF-κB target gene transcription in a reconstituted B cell system upon self-recognition of common BcR-intrinsic epitopes. However, the precise molecular details of such process are unknown. In order to gain insight into the molecular interactions, particularly to further understand the role played by autonomous signaling, we determined the crystal structures of two BcR IG of CLL cases assigned to subset #4. This is a CLL subset expressing stereotyped, G(κ)-switched BcR IG encoded by the IGHV4-34/IGKV2-30 gene combination. Subset #4 accounts for ~1% of all CLL and is the largest within M-CLL, distinctive for a particularly indolent clinical course. BcR IG derived from two subset #4 cases were found to bind autologously via their VH CDR3 loops to a composite surface spanning the variable and constant regions of the heavy chain; the relevant epitope is conserved in all cases belonging to subset #4 and differs from other non-subset #4 BcR IG. This specific self-recognition was identified as dependent on the individual IG gene usage in the BcR, and is functionally relevant as it occurs in solution and leads to intracellular signalling in B cells. Analysis of epitope and paratope mutants revealed that the interactions observed in the crystal structures are mediated by a few critical amino acid residues. Indeed, the distinctively conserved amino acid residues in the VH CDR3 loop of the BcR IG both dictate a specific VH-VK pairing and shape the combining site for autologous recognition. Moreover, the epitope comprises specific amino acids from the CH1 domain that restrict the autologous recognition to IgG molecules. Finally, we found persisting long-lived interaction occurring between subset #4 BcR IGs, thus recalling high affinity receptor-cognate antigen interactions associated with the induction of anergy. This scenario well fits with the anergic phenotype of the subset #4 leukemic cells, and thus provides a biochemical explanation for the indolent clinical course of this subset. In conclusion, though focusing on a particular CLL subset, the structural and biochemical analysis here presented describes a general model for autologous recognition that may epitomize the molecular events leading to the expansion of CLL B lymphocytes at large. It is conceivable that CLL-associated BcR IGs can each bind to a distinct internal epitope with the specific nature of the interaction dictated by diverse factors e.g. VDJ recombination, heavy and light chain pairing, SHM, and isotype switch. The strength and persistence of the autologous recognition can then lead to a specific outcome in the intracellular signaling process, ranging from proliferation to anergy. The structural diversity thus produced in the BcR IG development may be linked to and underlie the heterogeneity characterizing CLL at the biological and clinical level. Disclosures Stamatopoulos: Gilead Sciences: Research Funding; Janssen Pharmaceuticals: Research Funding. Ghia:Pharmacyclics: Honoraria; Gilead: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria; Roche: Research Funding; GSK: Research Funding; AbbVie: Honoraria; Celgene: Honoraria; Adaptive Biotechnologies: Consultancy.

A broad range of mutations in HIV-1 neutralizing human monoclonal antibodies specific for V2, V3, and the CD4 binding site

The HIV vaccine-induced neutralizing antibodies (Abs) display low rates of mutation in their variable regions. To determine the range of neutralization mediated by similar human monoclonal Abs (mAbs) but derived from unselected chronically HIV-1 infected subjects, we tested a panel of 66 mAbs specific to V3, CD4 binding site (CD4bs) and V2 regions. The mAbs were tested against 41 pseudoviruses, including 15 tier 1 and 26 tier 2, 3 viruses, showing that the neutralization potency and breadth of anti-V3 mAbs were significantly higher than those of the anti-CD4bs and anti-V2 mAbs, and only anti-V3 mAbs were able to neutralize some tier 2, 3 viruses. The percentage of mutations in the variable regions of the heavy (VH) and light (VL) chains varied broadly in a range from 2% to 18% and correlated moderately with the neutralization breadth of tier 2, 3 viruses. There was no correlation with neutralization of tier 1 viruses as some mAbs with low and high percentages of mutations neutralized the same number of viruses. The electrostatic interactions between anti-V3 mAbs and the charged V3 region may contribute to their neutralization because the isoelectric points of the VH CDR3 of 48 anti-V3 mAbs were inversely correlated with the neutralization breadth of tier 2, 3 viruses. The results demonstrate that infection-induced antibodies to CD4bs, V3 and V2 regions can mediate cross-clade neutralization despite low levels of mutations which can be achieved by HIV-1 vaccine-induced antibodies.

Charting Unique Signatures of Somatic Hypermutation Amongst Chronic Lymphocytic Leukemia Patients Expressing IGHV4-34 Clonotypic B Cell Receptors

The human IGHV4-34 gene encodes antibodies which are intrinsically autoreactive when the VH domain is unmutated. Therefore, B cells expressing IGHV4-34 B-cell receptor immunoglobulins (BcR IG) are normally under close scrutiny in order to avoid unwanted autoreactivity, especially against DNA. The IGHV4-34 gene is frequently utilized in chronic lymphocytic leukemia (CLL), where, typically, it shows a high load of somatic hypermutation (SHM). We have previously reported distinctive SHM patterns amongst IGHV4-34 CLL, especially for subsets with stereotyped BcR IG. However, although a large number of cases (~2000) was previously studied, since even the largest subsets account for only ~3% of CLL, meaningful conclusions could not be reached for smaller subsets. Here we revisit this issue in a series of 16,528 CLL cases and focus on IGHV4-34 expressing subsets: #4 (IGHV4-34/IGHD5-18/IGHJ6 | 156 cases, 0.9%); #11 (IGHV4-34/IGHD3-10/IGHJ4 | 16 cases, 0.1%); #16 (IGHV4-34/IGHD2-15/IGHJ6 | 41 cases, 0.25%); #29 (IGHV4-34/IGHD: unassignable/IGHJ3 | 39 cases, 0.24%); and #201 (IGHV4-34/IGHD: unassignable/IGHJ3 | 43 cases 0.26%). Focusing on codons 27-104 within the VH domain (from CDR1-IMGT to FR3-IMGT), we calculated the sequence distance between subsets and the corresponding IGHV4-34 germline sequence based on a pairwise qualitative and quantitative comparison of the respective amino acid composition. The minimum distance calculated, and hence the greatest identity, was observed between subsets #4 and #16, both concerning IgG-switched cases (IgG-CLL), which is notable given the overall rarity of IgG-CLL. In contrast, the maximum distance, implying the least identity, was between subsets #16 and #201, the latter concerning IgM/D-CLL. Extreme variations between subsets were noted in codons spanning the entire VH domain. This result is consistent with our finding of a subset-biased distribution of mutations over the VH domain. More specifically, while subsets #11, #16, #29 and #201 had a lower frequency of mutations within VH CDR1 compared to VH CDR2, the exact opposite was seen in subset #4, with 40% of mutations in VH CDR1 versus 27% in VH CDR2. In addition, subsets #4, #11, #16 and #29 had a similar distribution of mutations in VH FR2 and VH FR3, in contrast to subset #201 that showed a preference for VH FR3 over VH FR2. Consequently, we noted that certain positions were targeted in a subset-specific manner e.g. codon 28 in VH CDR1 was heavily targeted in subsets #4 (68.6%) and #16 (87.8%), with most cases carrying an acidic amino acid (AA) introduced by SHM, glycine to glutamic acid, G>E: 51.3% for subset #4 and 78% for subset #16. The high prevalence of acidic AA introduced by SHM in these subsets is notable considering the electropositive nature of their VH CDR3 (especially of subset #4), strongly recalling edited anti-DNA antibodies. Interestingly, the G>E change was identified at a much lower frequency in other IGHV4-34 subsets: 18.75% for subset #11; 2.6% for subset #29; 7% for subset #201, all of which carried electronegative VH CDR3. Further, we noted that certain positions were heavily targeted in all subsets e.g. 56-86% targeting for SHM at codon 92 in VH FR3 where serine is encoded by the agc triplet, the ”hottest of hotspots”. This result could be viewed as sequence- rather than subset-dependent and linked to the molecular features of this codon, which is supported by the low targeting of codon 93 (0-6%), also encoding serine by the tct triplet. Other positions were targeted in all subsets but at vastly different frequencies e.g. codon 64 was targeted in 37.8% in subset #4 rising to 100% in subset #29. Finally, positions heavily targeted by SHM in certain subsets were unmutated in other subsets e.g. codon 36 in VH CDR1 remained unmutated in subset #16, in contrast 76.9% of subset #29 were mutated at this position resulting in an AA change. In conclusion, we document different spectra of SHM and AA changes between stereotyped IGHV4-34 CLL subsets. The finding of subset-biased, recurrent AA changes at certain codons indicates that the respective progenitor cells may have responded in a specific manner to the selecting antigen(s), despite expressing the same IGHV gene, indicating a functional purpose for these modifications. This is exemplified by the molecular characteristics of the recurrent AA changes in subset #4, thereby offering interesting pathogenetic hints. DisclosuresNo relevant conflicts of interest to declare.

Engineered Bovine Antibodies in the Development of Novel Therapeutics, Immunomodulators and Vaccines

Some bovine antibodies across all classes are unique, such as the CDR3 of the variable heavy-domain (VH CDR3), which is exceptionally long (up to 66 amino acids), unlike most conventional antibodies where the VH CDR3 loops range from 10 to 25 amino acids. The exceptionally long VH CDR3 is encoded by unusually long germline IGHD genes together with insertion of novel “a” nucleotide rich conserved short nucleotide sequence (CSNS) specifically at the IGH V-D junction. Such an exceptionally long VH CDR3 confers unique “knob and stalk” structural architecture where the knob, formed by intra-VH CDR3 disulfide bridges, is separated by 20 Å solvent exposed stalk composed of anti-parallel beta strands. The substitution of the knob with cytokines, such as, erythropoietin and granulocyte colony stimulating factor 3 (granulocyte colony stimulating factor), results in expression of functional fusion proteins with enhanced pharmacokinetics. The beta stranded stalk can be substituted with other rigid structures, for example, repeat alpha helices to form coiled-coil that mimics the beta-stranded stalk and, thus, opens opportunities for insertion of this structure in the CDRs of antibodies across species. Given the versatility of such a structural platform in bovine antibody VH CDR3, it provides the opportunity for the development of new generation of diagnostics, therapeutics, vaccines and immunomodulating drugs.

The role of VH CDR3 in determining Tn‐antigen specificities of MLS128 and 83D4 monoclonal antibodies (1007.1)

Tn‐antigen (GalNAcα‐Ser/Thr) is associated with carcinomas. MLS128 and 83D4 are Tn‐antigen specific monoclonal antibodies. They bind carbohydrate epitopes consisting of three or two consecutive Tn‐antigens (Tn3 or Tn2) with different affinities. Since VH sequences of both mAbs are nearly identical except a few amino acids in the CDR3, the aim of this study was to evaluate the role of the VH CDR3 in determining their Tn‐antigen specificities.The VH domain of 83D4 was cloned into pCI‐MLS128 VL‐Fc to construct pCI/MLS128/83D4 VH CDR3 scFv‐Fc vector. CHO cells transfected with the vector were cultured in a selective medium to screen clones expressing MLS128/83D4 VH CDR3 scFv‐Fc protein. Fc‐levels in media of stable clones were determined by ELISA. Of several stable clones producing the MLS128/83D4 VH CDR3 scFv‐Fc protein at 2 µg/ml levels established, one clone was chosen for purification and characterization of the MLS128/83D4 VH CDR3 scFv‐Fc protein. From 330 ml of culture medium, 70 μg of the scFv‐Fc protein were purified to near homogeneity by Protein A‐Sepharose chromatography. Binding activity of three scFv‐Fc proteins (MLS128/83D4 VH CDR3, MLS128, 83D4) was compared by ELISA using Tn2‐ and Tn3 peptides.

Clonal heterogeneity of thymic B cells from early-onset myasthenia gravis patients with antibodies against the acetylcholine receptor

Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR–MG) is considered as a prototypic autoimmune disease. The thymus is important in the pathophysiology of the disease since thymus hyperplasia is a characteristic of early-onset AChR–MG and patients often improve after thymectomy. We hypothesized that thymic B cell and antibody repertoires of AChR–MG patients differ intrinsically from those of control individuals. Using immortalization with Epstein–Barr Virus and Toll-like receptor 9 activation, we isolated and characterized monoclonal B cell lines from 5 MG patients and 8 controls. Only 2 of 570 immortalized B cell clones from MG patients produced antibodies against the AChR (both clones were from the same patient), suggesting that AChR-specific B cells are not enriched in the thymus. Surprisingly, many B cell lines from both AChR–MG and control thymus samples displayed reactivity against striated muscle proteins. Striational antibodies were produced by 15% of B cell clones from AChR–MG versus 6% in control thymus. The IgVH gene sequence analysis showed remarkable similarities, concerning VH family gene distribution, mutation frequency and CDR3 composition, between B cells of AChR–MG patients and controls. MG patients showed clear evidence of clonal B cell expansion in contrast to controls. In this latter aspect, MG resembles multiple sclerosis and clinically isolated syndrome, but differs from systemic lupus erythematosus.Our results support an antigen driven immune response in the MG thymus, but the paucity of AChR-specific B cells, in combination with the observed polyclonal expansions suggest a more diverse immune response than expected.

Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity

In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.

Stereotyped subset #1 chronic lymphocytic leukemia: a direct link between B‐cell receptor structure, function, and patients' prognosis

Chronic lymphocytic leukemia (CLL) with stereotyped B-cell receptor (BCR) belonging to subset #1 (IGHV1-5-7/ IGKV1-39) display a poor outcome. To characterize their genetic and genomic features and BCR function, we selected 20 subset #1 CLL from a series of 579 cases. Subset #1 CLL, all showing unmutated IGHV, were associated with the presence of del(11q) (50%) in comparison with unmutated CLL, unmutated stereotyped CLL other than subset #1 and with cases using the same IGHV genes but a heterogeneous VH CDR3 (non-subset #1 CLL). There were no distinctive features regarding CD38, ZAP-70, and TP53 disruption. NOTCH1, SF3B1, and BIRC3 were mutated in 15%, 0%, and 5% of cases, respectively, while BIRC3 was deleted in 22% of cases. Microarray unsupervised analysis on 80 unmutated/mutated/stereotyped/non-stereotyped CLL showed a tight clustering of subset #1 cases. Their genomic signature exhibited several differentially expressed transcripts involved in BCR signal transduction, apoptosis regulation, cell proliferation, and oxidative processes, regardless of del(11q). Accordingly, BCR ligation with anti-IgM revealed a significant higher proliferation of subset #1 versus unmutated non-subset #1 CLL, both at baseline and after 24–48 hr stimulation. Subset #1 CLL represent a paradigmatic example of the direct link between BCR structure, function, and patients prognosis.

IgG variable region and VH CDR3 diversity in unimmunized mice analyzed by massively parallel sequencing

Most antigen-specific mouse antibodies have been derived by hybridoma technology, predominantly through use of the Balb/c strain. Much of the Balb/c germline repertoire of variable genes (V regions) is known. However, there is little information about the background expressed repertoire of IgG antibodies in mice, which reflects the baseline against which antigen-specific antibodies are generated through immunization. To assess this baseline repertoire, RNA was isolated from splenic B-cells enriched for expression of IgG from three mice. The RNA was individually amplified with three distinct PCR primer sets for comprehensive recovery of the heavy and light chain variable regions. Each PCR product was independently subjected to deep sequencing using 454 pyro-sequencing technology and analysed for redundancy, open reading frame, germline representation, and CDR3 sequence of the heavy chain variable region (VH CDR3) within and across the primer sets and mice. A highly skewed abundance of heavy and light chain variable gene usage was observed for all three primers in all three mice. While showing considerable overlap, there were differences among these profiles indicative of primer bias and animal-to-animal variation. VH CDR3 sequences were likewise highly skewed indicating that the heavy chain genes profiles substantially reflected individual antibodies. This observation was confirmed through analysis of randomly selected complete heavy chain variable sequences. However, there was very little redundancy in VH CDR3 sequences across the different mice. We conclude that the background IgG repertoire in young, unimmunized mice is highly skewed within individual mice and is diverse among them, a pattern similar to that observed in highly immunized mice.

Abstract 5478: A bispecific enediyne-energized fusion protein Ec-LDP-Hr-AE enhances anti-cancer efficacy on esophageal cancer cells by targeting EGFR and HER2.

Abstract Esophageal cancer is one of the most common cancers, and the 5-year survival rate is less than 10% due to lack of effective therapeutic agents. This study was to evaluate antitumor activity of Ec-LDP-Hr-AE, a recently developed bispecific enediyne-energized fusion protein, on esophageal carcinoma, targeting both EGFR (epidermal growth factor receptor) and HER2 (human epidermal growth factor receptor 2), because both EGFR and HER2 have been shown overexpression in over 30% of esophageal cancers. The fusion protein Ec-LDP-Hr-AE was encoded by a gene that was genetically constructed by fusing the oligopeptides ligand Ec (22 amino acids of EGF COOH-terminal) and Hr (VH CDR3 region of anti-HER2 C6.5 antibody) with lidamycin (LDM, an enediyne antibiotic with potent cytotoxicity) via two (G4S)3-linker. The encoded 18.4 kDa fusion protein was expressed in Escherichia coli and characterized its binding affinity with esophageal squamous cell carcinoma (ESCC) cells by flow-cytometry-based immunofluorescence assay. The results showed that Ec-LDP-Hr-AE protein bound to ESCC cells EC9706 and KYSE150 with high affinity (the Kd values were 5.283μmol/L and 3.562μmol/L, respectively). The potency of Ec-LDP-Hr-AE was determined on four human ESCC cell lines with differential expression of EGFR and HER2 using MTT assay. The bispecific Ec-LDP-Hr-AE showed potent cytotoxicity to EC1, EC109, EC9706 and KYSE150 cells with IC50 values in the picomolar level. Moreover, the bispecific Ec-LDP-Hr-AE was more potent than the monospecific counterparts (Ec-LDP-AE and LDP-Hr-AE) and LDM. Cell cycle distribution assay revealed that Ec-LDP-Hr-AE caused G2-M arrest in EC9706 and KYSE150 cells, in which the G2-M phase cells reached the peak with 0.1nmol/L of Ec-LDP-Hr-AE treatment. Ec-LDP-Hr-AE also induced cancer cell apoptosis in EC9706 and KYSE150 cells and the induction of apoptosis was in a dosage-dependent manner. The apoptotic index in the EC9706 cells exposed to 0.1, 0.5, or 1 nmol/L of Ec-LDP-Hr-AE were 15.0 ± 0.3%, 38.1 ± 0.6%, and 50.0 ± 0.4%, respectively. Western blotting showed that Ec-LDP-Hr-AE promoted caspase-3 and caspase-7 activities as well as PARP cleavage, indicating that Ec-LDP-Hr-AE-induced apoptosis was associated with mitochondria-caspase cascade. Moreover, Ec-LDP-Hr-AE inhibited KYSE150 cancer cell proliferation via decreasing phosphorylation of EGFR and HER2, and further exerted inhibition of the activation of their downstream signaling molecules (e.g. AKT and ERK). In summary, the bispecific energized fusion protein Ec-LDP-Hr-AE exhibited potent cytotoxicity to ESCC cells in vitro, leading to cell cycle arrest and apoptosis via targeting EGFR and HER2. These results suggest that Ec-LDP-Hr-AE could be a promising candidate for esophageal cancer targeting therapy. Citation Format: Guo Xiaofang, Xiaofei Zhu, Yue Shang, Shenghua Zhang, Yongsu Zhen, Wancai Yang. A bispecific enediyne-energized fusion protein Ec-LDP-Hr-AE enhances anti-cancer efficacy on esophageal cancer cells by targeting EGFR and HER2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5478. doi:10.1158/1538-7445.AM2013-5478

Validation of Stereotyped Immunoglobulin Heavy Chain CDR3 Sequences As Candidate Antigens for Immunotherapy of CLL

AbstractAbstract 1775The immunoglobulin gene repertoire in CLL is remarkably restricted with greater than 30% of cases carrying quasi-identical (stereotyped)heavy complementarity-determining region 3 (VH CDR3) sequences. Indeed, cases can be clustered into different subsets based on shared, subset-biased motifs within the clonotypic VH CDR3s, with, notably, only a handful of subsets accounting for almost 10% of all CLL. VH CDR3 stereotypes are more frequent in cases with unmutated IGHV genes (U-CLL) who are associated with adverse prognosis. In principle, VH CDR3 stereotypy might allow to exploit these IG motifs as candidate Tumor Associated Antigens (TAA) for targeted immunotherapy of CLL. The aim of our study was to validate as potential TAA subset-specific IG motifs from major CLL subsets, focusing especially on subsets #1 and #2 that are the largest overall and both associated with aggressive clinical course.We have so far identified, by in silico analysis, 1–3 long peptides (15-mer) encompassing the VH CDR3 protein regions of subsets #1, 2, 4, 6, 8, 10 with (i) high binding score to MHC class II molecules and (ii) also containing minimal HLA class I-specific epitopes (HLA-A2, -A3, -A24, DR1, DR7, DR13 that are most frequent in the Caucasian population).Blood lymphocytes from 18 CLL patients were collected and phenotyped by flow cytometry with appropriate antibodies to assess the expression of stimulatory, co-stimulatory and negative regulatory molecules on both T and B cells. In addition, HLA typing of CLL patients was performed to select patients expressing the aforementioned HLA molecules. Overall, 13/18 patients matched the defined HLA class I and/or class II molecules.Negatively purified T cells from 11 CLL patients expressing HLA-A2 and/or DR13 have been then stimulated in vitro with the synthesized peptides of the specific stereotype (subset #1 and 2) in the presence of culture medium containing 5% of human serum plus IL-2 (20 IU/ml) and IL-15 (10 ng/ml). These T lymphocytes were then weekly stimulated with autologous irradiated antigen presenting cells (APC; monocytes, B cells, etc.) pulsed with the peptides. Starting from the third week of culture, the specific recognition of CDR3-derived TAAs and of tumor cells (autologous CLL cells) by the T cell cultures has been assessed by in vitro functional assays (ELISPOT assay).We were able to isolate CDR3- (subsets #1 and #2) and tumor-specific T cells from 5/11 CLL patients. In addition, in 4 selected patients the Ag- and tumor specific T lymphocytes have been expanded in vitro by Rapid Expansion Protocol (REP), based on the stimulation of T cells with allogeneic irradiated PBMCs from healthy donors plus OKT3 and high doses of IL-2. Using this protocol we were able to obtain large numbers (2–10 ×109) of anti-CDR3 T cells in all 4 cases tested, thereby, in principle, achieving the potential to use this protocol for expanding sufficient cells for clinical applications. Interestingly, post-REP T cell cultures showed enrichment (85–90%) of CD3+CD8+ T cells and down-modulation of negative regulatory molecules, such as CTLA-4, as compared to pre-REP in vitro stimulated T cells. These cells could be expanded in vitro for up to 6 weeks without any decay in proliferation.Taken together, these results indicate that stereotyped VH CDR3 peptide sequences can represent candidate antigens to elicit T cell-mediated anti-CLL responses, especially in poor prognosis cases, where therapeutic innovation is more urgently needed. After validation of this protocol in a larger series, our results may provide the proof of principle for the design of new immunotherapy protocols for CLL, including both active vaccination and adoptive cell therapy. Disclosures:No relevant conflicts of interest to declare.

Molecular Characterization of Immunoglobulin Gene Rearrangements in Diffuse Large B-Cell Lymphoma: Antigen-Driven Origin and IGHV4-34 as a Particular Subgroup of the Non-GCB Subtype

The pathogenesis of diffuse large B-cell lymphoma (DLBCL) remains partially unknown. The analysis of the B-cell receptor of the malignant cells could contribute to a better understanding of the DLBCL biology. We studied the molecular features of the immunoglobulin heavy chain (IGH) rearrangements in 165 patients diagnosed with DLBCL not otherwise specified. Clonal IGH rearrangements were amplified according to the BIOMED-2 protocol and PCR products were sequenced directly. We also analyzed the criteria for stereotyped patterns in all complete IGHV-IGHD-IGHJ (V-D-J) sequences. Complete V-D-J rearrangements were identified in 130 of 165 patients. Most cases (89%) were highly mutated, but 12 sequences were truly unmutated or minimally mutated. Three genes, IGHV4-34, IGHV3-23, and IGHV4-39, accounted for one third of the whole cohort, including an overrepresentation of IGHV4-34 (15.5% overall). Interestingly, all IGHV4-34 rearrangements and all unmutated sequences belonged to the nongerminal center B-cell-like (non-GCB) subtype. Overall, we found three cases following the current criteria for stereotyped heavy chain VH CDR3 sequences, two of them belonging to subsets previously described in CLL. IGHV gene repertoire is remarkably biased, implying an antigen-driven origin in DLBCL. The particular features in the sequence of the immunoglobulins suggest the existence of particular subgroups within the non-GCB subtype.

Mutational Status and Gene Repertoire of IGHV-IGHD-IGHJ Rearrangements in Serbian Patients With Chronic Lymphocytic Leukemia

The mutational status and configuration of immunoglobulin heavy variable (IGHV) gene rearrangements was analyzed in 85 Serbian patients with chronic lymphocytic leukemia (CLL). We found that 55.3% of cases belonged to mutated and 44.7% to unmutated CLL, progressive disease predominating in the unmutated subset. IGHV gene use resembled that obtained for Mediterranean countries, except for underrepresentation of the IGHV4 subgroup in our cohort. Chronic lymphocytic leukemia (CLL) results from the clonal expansion of mature B lymphocytes and is characterized by extreme clinical heterogeneity. One of the most reliable prognostic markers in chronic lymphocytic leukemia (CLL) is the mutational status of immunoglobulin heavy variable (IGHV) genes, which defines 2 subsets, mutated CLL (M-CLL) and unmutated CLL (U-CLL), with different clinical courses. Biased IGHV gene use between M-CLL and U-CLL clones, as well as population differences in the IGHV gene repertoire have been reported. In this study, mutational status and configuration of IGHV-IGHD-IGHJ rearrangements in 85 Serbian patients were analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing methodology. We found that 55.3% of cases belonged to M-CLL and 44.7% belonged to U-CLL, with progressive disease predominating in the unmutated subset. Most frequently expressed was the IGHV3 subgroup (55.7%), followed by IGHV1 (27.3%), IGHV4 (12.5%), IGHV5 (2.3%), IGHV2 (1.1%), and IGHV6 (1.1%). The distribution of IGHD subgroups was as follows: IGHD3, 39.1%; IGHD2, 21.8%; IGHD6, 12.6%; IGHD1, 10.3%; IGHD4, 8%; IGHD5, 6.9%; and IGHD7, 1.1%. The most frequent IGHJ gene was IGHJ4 (48.9%), followed by IGHJ6 (28.4%), IGHJ3 (11.4%), and IGHJ5 (11.4%). In 15.3% of cases, heavy complementarity-determining region 3 (VH CDR3) amino acid sequences could be assigned to previously defined stereotyped clusters. Our study showed a strong correlation between IGHV gene mutational status and clinical course of CLL. IGHV gene use was comparable to that obtained for Mediterranean countries, with the exception of the IGHV4 subgroup, which was underrepresented in our cohort.

Analysis of SF3B1 mutations in monoclonal B‐cell lymphocytosis

Analysis of <i>SF3B1</i> mutations in monoclonal B‐cell lymphocytosis

Distinctive Patterns of Intraclonal Diversification In IGHV1-2*04 Immunoglobulin Receptors of Patients with Splenic Marginal Zone Lymphoma: A of Ongoing Interactions with Antigen?

Abstract 2638We recently demonstrated that over 30% of cases with splenic marginal-zone lymphoma (SMZL) express distinctive immunoglobulin (IG) receptors that utilize a single polymorphic variant of the IGHV1-2 gene (IGHV1-2*04) and also exhibit restricted antigen-binding site motifs and precise targeting of somatic hypermutation (SHM). On these grounds, we proposed the existence of molecular subtypes of SMZL defined by immunogenetic analysis of the IG receptors with implications for selection by specific (super) antigenic element(s) in the development of at least a major subset of SMZL. In order to gain insight as to whether antigen involvement is relevant only prior to the malignant transformation or if it continues to affect the SMZL clone itself leading to intraclonal diversification (ID) through ongoing SHM, we conducted a large-scale subcloning study of rearranged IG heavy variable genes, in a total of 471 subcloned sequences from 22 SMZL cases. The analysis was intentionally biased towards cases expressing IGHV1-2*04 receptors that exhibit a series of distinctive immunogenetic features, including biased usage of the IGHD3-3 and IGHD3-10 genes, unusually long heavy complementarity-determining region 3 (VH CDR3) and minimally/borderline mutated status (identity to the germline in the range of 97–99.6%). Hence, the study group included 16 IGHV1-2*04 cases and 6 cases utilizing other IGHV genes. PCR reactions were run using the high-fidelity Accuprime Pfx polymerase and a median of 21 (9–46) colonies/case were analyzed. All “non-ubiquitous” sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample; (ii) confirmed mutation (CM) - a mutation observed in more than one but in less than all subcloned sequences from the same sample. Overall, 15/22 cases (68%) carried intraclonally diversified IGHV-D-J genes with CMs amongst subclones, of which 12 utilized the IGHV1-2*04 gene whereas the remaining 3 utilized other IGHV genes. The high frequency of ID within the IGHV1-2*04 group, ranging from limited to (often) pronounced, is noteworthy in view of the generally low level of SHM among IGHV1-2*04 receptors. Detailed analysis of the distribution and molecular features of CMs revealed: (i) restricted ID patterns, in the sense of identical mutations in certain VH positions among subclones of different cases; (ii) “hotspots” of ID, i.e. particular codons exhibiting intense ongoing mutational activity (especially notable in this respect was codon 39 in VH FR2); (iii) a predominance of conservative amino acid changes, characterized by somatically introduced amino acid belonging to the same biochemical category as the mutating amino acid; and (iv) limited diversification within VH CDR3. Additionally, in 7/12 IGHV1-2*04 cases with CMs, identified ID patterns delineate distinct “clusters” of subcloned sequences with unique as well as shared mutations and closely similar if not identical VH CDR3s (including identical VH CDR3 length), pointing to “branching” of the malignant clone into distinct subclones, perhaps able to evolve along related yet distinct pathways. In conclusion, our study indicates that the SHM mechanism may continuously operate in certain subsets of SMZL, especially those expressing IGHV1-2*04 receptors. Although the precise timing of interactions with antigen(s) and their functional implications for SMZL evolution will likely remain difficult to define accurately, the results reported here suggest a role for persistent antigenic stimulation, at least for certain immunogenetically defined subsets of SMZL. Disclosures:Mollejo:Red Temática de Investigación Cooperativa en Cancer (RETICC): Research Funding; Asociación Española contra el Cancer: Research Funding.

Primary Intraocular Lymphomas Display A Remarkably Biased Immunoglobulin Heavy Chain Gene Repertoire and Precisely Targeted Somatic Hypermutation Suggesting Antigenic Selection of the Neoplastic Cells

Abstract 1574Primary intraocular lymphoma (PIOL) is a high grade lymphoma, which affects the retina, vitreous and/or the optic nerve. The vast majority of cases are classified as diffuse large B cell lymphoma and considered as a subtype of primary central nervous system lymphoma (PCNSL). Because of its rarity, only very few studies have been performed on immunoglobulin (IG) gene repertoire of PIOL mostly in very small series. We here report results from the detailed immunogenetic analysis of the clonal IGHV-IGHD-IGHJ rearrangements in 30 cases of PIOL, the largest series to date. We observed a highly restricted IGHV usage, with a single gene, namely IGHV4-34, present in two thirds of cases (20/30). The second most frequent gene was IGHV3-7, utilized in 10% (3/30) of cases. Thus, only two IGHV genes accounted for more than three quarters of the PIOL repertoire. IGHD3 and IGHD2 subgroup genes were by far the two most frequently used, respectively in 51.9% (14/30) and 27.9% (7/30) of cases. In contrast, the IGHD6 subgroup genes, commonly used in other B cell malignancies, were not detected in any PIOL IGH rearrangement. While IGHJ4 was found in 40% (12/30) cases, IGHJ usage was unusual for the IGHJ5 and IGHJ6 genes, as the former clearly predominated over the latter (10/30 cases, 33.3% vs 4/30 cases, 13.3%). Heavy complementarity-determining region 3 (VH CDR3) length ranged from 7 up to 27 amino acids (median, 14). Twenty of 30 cases carried electropositive VH CDR3s with predicted isoelectric values of 7.0 or greater (up to 13.0). Remarkably, all 3 cases expressing the IGHV3-7 gene had 11 aminoacid-long, electropositive VH CDR3s with shared motifs, enabling their assignment to a cluster with “stereotyped” antigen-binding sites. Except for two unmutated cases, all sequences had a high number of somatic mutations as the mean % of identity from their germline counterpart was 85.9% (range 77.7% to 95.5%). Analysis of the distribution of somatic hypermutation (SHM) in the subgroup of PIOL cases utilizing the IGHV4-34 gene revealed high replacement (R) to silent (S) mutation ratios in CDRs (R/S>3.0) along with low R/S ratios in FRs. Shared replacement mutations (“stereotyped” amino acid (AA) changes) at certain codon positions were identified amongst rearrangements utilizing the IGHV4-34 gene, alluding to an antigen-driven SHM process. Several AA changes identified here were distinct from those previously reported for IGHV4-34 rearrangements in other B cell malignancies, in particular chronic lymphocytic leukemia, mantle-cell lymphoma or Burkitt’s lymphoma, and thus can be considered as “PIOL-biased”. In addition, the IGVH4-34 specific motif responsible for binding in superantigenic fashion the N-acetyllactosamine antigenic determinant was altered in only 2/20 IGVH4-34 PIOL sequences; however, the most critical residue of this motif (TRP at position 7 in FR1) was intact in all 20 cases. On these grounds, it could be hypothesized that despite intense SHM activity, these clones retained the ability to bind to and be activated by superantigens. Sequencing multiple clones (13 to 17) in three cases demonstrated intraclonal diversity in all of them, indicating ongoing mutational activity. In conclusion PIOL displays a highly biased IG gene repertoire with very precise targeting and distinctive features of SHM, suggestive of selection by specific (super)antigen(s) in lymphomagenesis. Disclosures:No relevant conflicts of interest to declare.