scholarly journals Frequency and Epitope Specificity of Anti-Factor VIII C1 Domain Antibodies in Acquired and Hereditary Hemophilia Inhibitor Patient Plasma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3491-3491 ◽  
Author(s):  
Joerg Kahle ◽  
Aleksander Orlowski ◽  
Anja Schmidt ◽  
Kerstin Brettschneider ◽  
Thomas Klingebiel ◽  
...  
Keyword(s):  
Amino Acid ◽  
Research Funding ◽  
Hemophilia A ◽  
Antibody Binding ◽  
Amino Acid Residues ◽  
C2 Domains ◽  
Epitope Specificity ◽  
C1 Domain ◽  
Competition Binding ◽  
Inhibitor Patient

Abstract Acquired hemophilia A (AHA) is a rare autoimmune disease caused by development of inhibitory anti-factor VIII (fVIII) antibodies (also called inhibitors) resulting in severe hemorrhages. In addition, inhibitor development is the most serious complication of today's replacement therapy in patients with hereditary X-linked hemophilia A (HA) disorder. Earlier studies showed that antibodies in AHA and HA inhibitor plasmas are both primarily directed to the A2 and C2 domains suggesting that these two domains are the predominant immunogenic fVIII regions (Fulcher et al, 1987; Prescott et al, 1997; Lollar, 2004). However, the C1 domain also makes a major contribution to the humoral anti-fVIII immune response in hemophilic mice (Healey et al, 2007), which motivated us to analyze the frequency and epitope specificity of anti-C1 antibodies in AHA and HA inhibitor patient plasma. The frequency of domain-specific antibodies were studied by antibody binding to human A2, C1 and C2 domains presented as (i) single human domain (SHD) human/porcine hybrid fVIII and (ii) HSA-fusion proteins. While similar frequencies of A2- and C2-specific antibodies were observed for both applied mapping strategies the use of isolated C1 domain resulted in much higher detection level of anti-C1 antibodies compared to the use of the human C1 domain human/porcine hybrid fVIII protein. As homologue-scanning mutagenesis relies on differences among human and porcine sequences these results suggest the presence of a large number of cross-reactive anti-C1 antibodies binding to species-conserved epitopes. Overall, anti-C1 antibodies were detected in 90 of 115 (78%) AHA and 36 of 63 (57%) HA inhibitor patients. Two well-characterized monoclonal C1 inhibitors, human LE2E9 (Jacquemin et al, 2000) and murine MAb 2A9 (ASH 2014 poster, Batsuli et al) were used for indirect epitope mapping of anti-C1 antibodies in AHA patients by competition binding studies. Our results for AHA patients with non-crossreactive anti-C1 antibodies only (n=11) show that antibody binding to human C1 domain human/porcine hybrid fVIII (HP53) protein was completely blocked in the presence of MAb 2A9. In contrast, antibody binding to the isolated C1 domain was only partially reduced in the presence of MAb 2A9 for a selected number of (high responding) AHA patients (n=10) suggesting the presence of a second population of crossreactive anti-C1 antibodies that exclusively bind to conserved amino acid residues. Competition binding to native and denatured fVIII and HP53 proteins revealed that MAb 2A9 and LE2E9 bind mutually exclusive to a conformational C1 epitope involving amino acid residues that are not conserved between humans and pigs. Consequently, essential binding residues were identified for both C1 inhibitors via the use of HP53 variants, in which surface exposed non-conserved amino acid residues on the human C1 domain were substituted for porcine residues. The results of this mutational analysis showed that despite their competitive binding different amino acid residues are essential for binding of MAb 2A9 and LE2E9. These findings are in agreement with the different specific inhibitory activities of the two C1 inhibitors (97 BU/mg vs 10000 BU/mg). Finally, HSA-C1 point mutants were used to directly map essential epitope residues of anti-C1 antibodies in AHA and HA inhibitor patient plasma. Our study demonstrates that a large number of AHA and HA inhibitor patients (126 of 178; 71%) have anti-C1 antibodies that comprise at least two different populations, crossreactive and non-crossreactive to porcine fVIII. Therefore, in addition to the A2 and C2 domains, the C1 domain seems to significantly contribute to the immune response to fVIII in these patients. As recent data point toward a functional role of the fVIII C1 domain for membrane-, fX-, and von Willebrand factor-binding (Lü et al, 2011) the clinical relevance of anti-C1 antibodies should be analyzed in further studies. Disclosures Tiede: Leo Pharma: Consultancy, Honoraria; Novo Nordisk: Consultancy, Honoraria, Research Funding; Coachrom: Research Funding; SOBI: Consultancy, Honoraria; Biogen Idec: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Boehringer Ingelheim: Consultancy, Honoraria; Octapharma: Other: Investigator, Speakers Bureau; Biotest: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Investigator, Research Funding; Baxter: Consultancy, Honoraria, Research Funding. Königs:Bayer: Research Funding, Speakers Bureau; Biotest: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Sobi: Consultancy; CSL Behring: Research Funding, Speakers Bureau; Intersero: Research Funding; NovoNordisk: Speakers Bureau.

Identification of Amino Acid Residues in the C1 Domain of Human Factor Va2 That Affect Phosphatidylserine-Triggered Cofactor Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1711-1711
Author(s):  
Rinku Majumder ◽  
Mary Ann Quinn-Allen ◽  
Barry R. Lentz ◽  
William H. Kane
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Factor Xa ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Hydrophobic Residues ◽  
Factor Va ◽  
C1 Domain ◽  
Cofactor Activity ◽  
Prothrombin Activation

Abstract Tightly associated factors Va and Xa serve as the essential prothrombin-activating complex whose assembly is triggered by occupancy of phosphatidylserine (PS) regulatory sites on both proteins. Factor Va C2 domain contains a binding site for soluble, short chain PS (C6PS) that includes the indole moieties of Trp2063/Trp2064 at the apex of a loop (“spike-1”) (Srivastava A, Quinn-Allen MA, Kim SW, Kane WH, Lentz BR. Biochemistry, 2001, 40(28): 8246–55). Our recent data show that there is a C6PS site in the factor Va2 C1 domain that serves as a regulatory site for assembly and/or activity of the FVa2-FXa complex (Majumder R, Quinn-Allen MA, Kane WH & Lentz BR. Manuscript in Preparation). This C6PS-binding site also involves aromatic and hydrophobic residues (Tyr1956/Tyr1957) located in a homologous loop whose apex is termed “spike 3”. In order to identify the amino acid residues in the C1 domain that contribute to the PS-mediated cofactor activity of factor Va2, charged and hydrophobic residues predicted to be exposed in FVa2-C1 domain were mutated to alanine in clusters of 1–3 mutations per construct. The resultant 20 mutants (R1880A, D1892A, (K1896,E1899)A, (F1900,L1901,Y1903)A, (E1905,R1907)A, Y1917A, (E1923,K1924)A, (K1941,E1942)A, (K1954,H1955)A, (Y1956,L1957)A, Y1956A, L1957A, K1958A, E1964A, K1980A, D1995A, R2019A, (R2023,R2027)A, R2023A, R2027A,) and factor V wild type were expressed in Cos-7 cells followed by activation with thrombin, partial purification and concentration using HiTrap SP HP columns. The specific activities of all factor Va mutants were greater than 70% of wild type, with concentrations in the 1.5-7μM range. Recently it has been shown that two mutants (Y1956, L1957)A and (R2023,R2027)A showed decreased binding to immobilized PS and a selective decrease in prothrombinase activity on membranes containing 5% PS (Saleh M, Peng W, Quinn-Allen MA, Macedo-Ribeiro S, Fuentes-Prior P, Bode W & Kane WH. Thromb. Haemost.2004, 91:16–27). Here we report the rate of prothrombin activation in the presence of 1 nM factor Xa, 5 nM factorVa2 (mutants and wild type) and 400 mM C6PS. Enhancement of cofactor activity (E) of factor Va-C1 wild type and mutants by C6PS was measured using the following equation ( Zhai X, Srivastava A, Drummond DC, Daleke D and Lentz BR. Biochemistry. 2002, 41: 5675–84): \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[E=\ (r_{Xa.Va.PL}/r_{Xa.PL})/(r_{Xa.Va}/r_{Xa})\] \end{document} Here, rXa·Va·PL is the rate of prothrombin activation measured as the initial slope of the rate of change of normalized DAPA fluorescence with time by enzyme in the presence of factor Va and lipid, and other terms are defined analogously. The cofactor activities of (Y1956, L1957) A, Y1956A and L1957A were drastically reduced (values are 1.1, 4.2 and 5.1 respectively) relative to the cofactor activity of the wild type factor Va2 (15). The cofactor activities of (R2023, R2027) A, E1964A and (K1954, H1955) A were also reduced but to a lesser extent (values are 8, 10.6 and 12 respectively). We plan to monitor the binding of these mutants to C6PS and to factor Xa in the presence of C6PS in order to determine the role of these mutations on the assembly and activity of prothrombinase. Supported by grants from the NHLBI (HL43106 to W. Kane and HL 072827 to BRL).

scholarly journals Identification of Amino Acid Residues in CD81 Critical for Interaction with Hepatitis C Virus Envelope Glycoprotein E2

2000 ◽  
Vol 74 (8) ◽  
pp. 3642-3649 ◽  
Author(s):  
Adrian Higginbottom ◽  
Elizabeth R. Quinn ◽  
Chiung-Chi Kuo ◽  
Mike Flint ◽  
Louise H. Wilson ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Envelope Glycoprotein ◽  
Tertiary Structure ◽  
Antibody Binding ◽  
Viral Envelope ◽  
Amino Acid Residues ◽  
Virus Envelope ◽  
Glycoprotein E2

ABSTRACT Human CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2. The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2. We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells. We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein. This residue was also important for binding of certain anti-CD81 monoclonal antibodies. Mutating residues 188 and 196 did not affect E2 or antibody binding. Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability. These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s).

scholarly journals The cDNA and derived amino acid sequence of porcine factor VIII

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4209-4214 ◽  
Author(s):  
JF Healey ◽  
IM Lubin ◽  
P Lollar
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Factor Viii ◽  
Untranslated Region ◽  
Hemophilia A ◽  
Sequence Determination ◽  
C2 Domains ◽  
Protein Alignments ◽  
A2 Domain

The cDNA corresponding to 137 bp of the 5′ untranslated region, the signal peptide, and the A1, A3, C1, and C2 domains of porcine factor VIII (fVIII) have been cloned and sequenced. Along with previously determined sequences of the porcine fVIII B domain and the A2 domain, this completes the sequence determination of the cDNA corresponding to the translated protein. Alignments of the derived amino acid sequence of porcine fVIII with human and murine fVIII indicate that the A1, A2, A3, C1, and C2 domains are more conserved than the B domains or the proteolytic cleavage peptides corresponding to residues 337–372 and 1649–1689. The knowledge of the porcine fVIII cDNA may be useful to understand functional and immunological differences between human and porcine fVIII and may lead to improved fVIII replacement products for hemophilia. A patients through the development of recombinant porcine fVIII or hybrid human/porcine fVIII derivatives.

scholarly journals The Frequency and Effect of Baseline Cross-Reacting and De Novo Inhibitors to Recombinant Porcine FVIII in Patients with Congenital and Acquired Hemophilia a

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1128-1128
Author(s):  
Carolyne Elbaz ◽  
Katerina Pavenski ◽  
Hina Chaudhry ◽  
Jerome M. Teitel ◽  
Michelle Sholzberg
Keyword(s):  
Factor Viii ◽  
Research Funding ◽  
Hemophilia A ◽  
De Novo ◽  
Factor Viia ◽  
Case Series ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
C2 Domains ◽  
Inhibitor Development

Background Patients with severe congenital hemophilia A (CHA) have a 25-40% lifetime risk of alloantibody (inhibitor) development to FVIII. Patients with acquired hemophilia A (AHA) spontaneously develop neutralizing autoantibodies to factor VIII. In both cases, patients require pro-hemostatic therapy with bypassing agents: recombinant factor VIIa (rFVIIa), activated prothrombin complex concentrate (aPCC) and more recently recombinant porcine factor VIII (rpFVIII). Anti-human FVIII (hFVIII) inhibitors typically bind to the A2 and C2 domains of the FVIII molecule. RpFVIII is an effective pro-hemostatic treatment for AHA and CHA given the immunologic difference in the A2 and C2 domains of the rpFVIII while maintaining sufficient hFVIII homology to act as an effective cofactor to human FIX in the intrinsic tenase. However, some anti-hFVIII antibodies cross-react with rpFVIII and may interfere with its hemostatic function. Cross-reacting antibodies were reported in 35% of subjects in a phase II/III trial prior to initiation of rpFVIII. Moreover, de novo rpFVIII inhibitors may develop during or after the treatment with rpFVIII and may affect its hemostatic function. Here we describe the largest case series to date on baseline cross-reactivity of rpFVIII inhibitors and post-treatment de novo inhibitor development in patients with CHA and AHA to address the paucity of published literature in this area. Aim First, we describe the frequency of baseline cross-reacting rpFVIII inhibitors in patients with AHA and CHA (with inhibitors) at our institution. Second, we describe the effect of baseline rpFVIII antibodies on FVIII recovery after treatment with rpFVIII. We also describe the frequency and timing of de novo rpFVIII inhibitor development after exposure to rpFVIII. Methods Institutional research ethics board approval was obtained. Electronic charts of patients admitted to our institution with AHA or CHA who underwent testing for rpFVIII inhibitors were reviewed retrospectively. RpFVIII inhibitor assay is performed in the special coagulation laboratory using the Nijmegen modified Bethesda assay. The patient sample is initially heat-treated at 57 Results Twenty-seven patients (7 CHA, 20 AHA) underwent testing for porcine inhibitors since assay availability in 2016. 61% (5/7 CHA, 11/20 AHA) of patients had a detectable rpFVIII inhibitor prior to exposure to rpFVIII; median titer 1.6 BU/ml (range 0.6-192). Eight patients with AHA with baseline cross-reacting inhibitors received rpFVIII. Of those, three achieved an initial FVIII recovery beyond 100% (132%, 148% and 177%) after approximately 100U/kg of rpFVIII and all three had very low anti-rpFVIII Bethesda titers (0.70, 0.85 and 0.9 BU/ml). Five patients did not achieve a FVIII recovery above 50% (46%, 46%, 40%, 36% and 0%) despite approximately 100U/kg of rpFVIII. Most patients who received rpFVIII were tested weekly for the duration of their treatment or hospital stay. Upon discharge, patients who were seen in clinic for follow up were tested for anti-hFVIII and anti-rpFVIII. Two AHA patients without a baseline inhibitor who received rpFVIII treatment developed a de novo inhibitor after 20 days (1 BU/ml) and 133 days (12 BU/ml), respectively. One AHA patient had a rise in baseline anti-rpFVIII titer after exposure to rpFVIII. Conclusion In conclusion, we found that 61% of patients with AHA and CHA tested for rpFVIII inhibitors had a detectable baseline cross-reacting inhibitor which is higher than previously described. Of those patients with a baseline inhibitor treated with rpFVIII, only 37.5% of patients had an appropriate rise in FVIII. Finally, 13% of patients without baseline inhibitors developed a de novo inhibitor after exposure to rpFVIII, an incidence comparable to previously published findings. Disclosures Pavenski: Bioverativ: Research Funding; Alexion: Honoraria, Research Funding; Octapharma: Research Funding; Shire: Honoraria; Ablynx: Honoraria, Research Funding. Teitel:BioMarin: Consultancy; CSL Behring: Consultancy; Octapharma: Consultancy; Novo Nordisk: Consultancy; Shire: Consultancy; Pfizer: Consultancy, Research Funding; Bayer: Consultancy, Research Funding. Sholzberg:Takeda: Honoraria, Research Funding; Baxter: Honoraria, Research Funding; Baxalta: Honoraria, Research Funding. OffLabel Disclosure: Recombinant porcine factor VIII is used to treated patients with congenital hemophilia A with allo inhibitors

B-Cell Epitopes Within The A2 and C2 Domains Of Coagulant Factor VIII Refined By Orthologous Protein Antigenicity

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1101-1101
Author(s):  
Philip M Zakas ◽  
Kristine Vanijcharoenkarn ◽  
Rebecca Markovitz ◽  
Shannon L. Meeks ◽  
Christopher B Doering
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
B Cell ◽  
Hemophilia A ◽  
Cross Reactivity ◽  
C2 Domain ◽  
B Cell Epitopes ◽  
C2 Domains ◽  
Group A ◽  
A2 Domain

Abstract Approximately 20-30% of severe hemophilia A patients develop inhibitors (anti-fVIII antibodies) to human fVIII (hfVIII) with the primary targets being the A2 and C2 domains. Studies of A2 domain-specific murine monoclonal antibodies (MAbs) targeting hfVIII identified 8 distinct groups of inhibitory MAbs displaying varying inhibitor titers and kinetics, with groups A, D, and E possessing the greatest inhibitory activity. Furthermore within the C2 domain, we previously characterized 5 distinct groups, whereby group BC antibodies were the most common and displayed the highest specific inhibitory activity. At present, a recombinant porcine fVIII (pfVIII) product (OBI-1, Baxter International) is being investigated within a pivotal clinical trial for persons with acquired hemophilia A. The rational for a recombinant pfVIII product stems from the limited cross reactivity of pfVIII, and potentially other fVIII orthologs, with antibodies developed against hfVIII and the prior clinical success of plasma-derived pfVIII. We have described the biochemical characterization of both pfVIII and, more recently, ovine fVIII (ofVIII) and now investigate their antigenic properties to define/refine key inhibitory epitopes and determine the amino acid differences that confer reduced antigenicity. Using an ELISA-based assay, 15 A2 and 12 C2 MAbs spanning all inhibitory groups were screened against B-domain deleted (BDD) pfVIII and ofVIII. Only 4 A2 domain targeting MAbs representing groups B, C, DE, and E were found to bind pfVIII and all displayed reduced cross-reactivity (Figure 1). Additionally, 3 MAbs within groups B, C, or E also bound ofVIII. No group A or D MAbs screened bound either ortholog. Of the C2 domain targeting MAbs tested, 3 MAbs (I109, D102, and ESH-8) from groups AB, B and C, respectively, bound pfVIII to lesser degrees. Nine of the 12 C2 targeting MAbs bound ofVIII, albeit at reduced signal strength, suggesting divergence in C2 epitopes between pfVIII and hfVIII as well as between pfVIII and ofVIII. Using these data and previous literature defining the specific epitopes of these MAbs, we suggest that species-specific amino acid sequence differences may be responsible for the differential binding. MAb413, a well characterized A2 group A inhibitor, was found not to bind either ortholog. Alanine scanning mutagenesis within the 484 – 508 epitope demonstrated that single point mutations at R484, Y487, R489, or P492 could each inhibit binding of MAb413 to below 10%. In addition, P485 or S488 mutations each reduced binding to approximately 40% (Lubin et al J Biol Chem 272:48). Both pfVIII and ofVIII share substitutions R484S, Y487H, R489G, and pfVIII contains unique substitutions P485A, S488P, and P492L. Since all but two A2 group A MAbs have been mapped to residues 484-508, and because all group A MAbs compete with each other for their epitope, we hypothesize that these substitutions are responsible for diminished binding of group A MAbs. Group D inhibitory antibodies have been mapped to residues 604-740. Within this region, there are 12 shared amino acid substitutions and 7 amino acids altered in either ortholog but not both. As it was demonstrated that the orthologs display differential cross reactivity, this suggests disparate amino acids alter inhibitor-epitope recognition. Walter et al. (J Biol Chem 2013 288:14) used small angle x-ray scattering (SAXS) to isolate the epitope of MAb 3E6, a C2 MAb group A inhibitor, to exposed loops R2209-S2216 and L2178-D2187. Our data show that this MAb is not cross reactive and both orthologs contain S2216T and S2182N substitutions. Finally, ESH-8, a well-studied C2 group C MAb, binds ofVIII and pfVIII equivalently at 29% of the hfVIII level. The epitope for ESH-8 contains amino acids 2248-2285 and within this region there is only one conserved substitution, F2275L. Therefore, we predict that substitution of this residue could restore or further diminish ESH-8 binding. These data provide evidence that the lack of A2 group A and D epitopes, as well as the C2 domain group BC epitopes within these orthologs may be responsible for the residual procoagulant activity in the context of inhibitor plasma. Furthermore, these data also suggest that 1) B-cell epitopes can be defined or refined through this approach, 2) less antigenic residues can be identified and 3) the information obtained can be utilized to rationally design recombinant fVIII products with reduced antigenicity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification of a F.VIII epitope recognized by a human hemophilic inhibitor

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 497-499 ◽  
Author(s):  
BC Lubahn ◽  
J Ware ◽  
DW Stafford ◽  
HM Reisner
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Factor Viii ◽  
Fusion Proteins ◽  
Cleavage Site ◽  
Hemophilia A ◽  
Antibody Binding ◽  
Bleeding Disorders ◽  
Enzymatic Cleavage ◽  
Coagulant Activity

Abstract Hemophilia A, one of the most common of the inherited bleeding disorders, results from a deficiency or abnormality of factor VIII (F.VIII). In approximately 15% of persons with hemophilia, treatment with exogenous F.VIII is complicated by the development of anti-F.VIII antibodies which block F.VIII coagulant activity. These antibodies have been termed inhibitors. To localize epitopes recognized by inhibitors, we used a lambda gt11 library which expresses small random fragments of F.VIII as fusion proteins. One epitope has been mapped to the 25-amino acid sequence lys-338 through asp-362 of F.VIII (E338–362). Immunoaffinity-purified antibodies that react with this epitope neutralize F.VIII:C activity. E338–362 is adjacent to an enzymatic cleavage site at arg-372 which is important in F.VIII activation. Hence, an antibody binding to E338–362 would probably block this cleavage and thereby block activation of F.VIII.

scholarly journals Identification of a F.VIII epitope recognized by a human hemophilic inhibitor

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 497-499
Author(s):  
BC Lubahn ◽  
J Ware ◽  
DW Stafford ◽  
HM Reisner
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Factor Viii ◽  
Fusion Proteins ◽  
Cleavage Site ◽  
Hemophilia A ◽  
Antibody Binding ◽  
Bleeding Disorders ◽  
Enzymatic Cleavage ◽  
Coagulant Activity

Hemophilia A, one of the most common of the inherited bleeding disorders, results from a deficiency or abnormality of factor VIII (F.VIII). In approximately 15% of persons with hemophilia, treatment with exogenous F.VIII is complicated by the development of anti-F.VIII antibodies which block F.VIII coagulant activity. These antibodies have been termed inhibitors. To localize epitopes recognized by inhibitors, we used a lambda gt11 library which expresses small random fragments of F.VIII as fusion proteins. One epitope has been mapped to the 25-amino acid sequence lys-338 through asp-362 of F.VIII (E338–362). Immunoaffinity-purified antibodies that react with this epitope neutralize F.VIII:C activity. E338–362 is adjacent to an enzymatic cleavage site at arg-372 which is important in F.VIII activation. Hence, an antibody binding to E338–362 would probably block this cleavage and thereby block activation of F.VIII.

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

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4129-4129
Author(s):  
Maria Gounari ◽  
Claudia Minici ◽  
Marcus Dühren-von Minden ◽  
Schneider Dunja ◽  
Alkhatib Alabbas ◽  
...  
Keyword(s):  
Amino Acid ◽  
B Cell ◽  
Research Funding ◽  
Clinical Course ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Amino Acid Residues ◽  
Self Recognition

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.

scholarly journals The cDNA and derived amino acid sequence of porcine factor VIII

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4209-4214 ◽  
Author(s):  
JF Healey ◽  
IM Lubin ◽  
P Lollar
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Factor Viii ◽  
Untranslated Region ◽  
Hemophilia A ◽  
Sequence Determination ◽  
C2 Domains ◽  
Protein Alignments ◽  
A2 Domain

Abstract The cDNA corresponding to 137 bp of the 5′ untranslated region, the signal peptide, and the A1, A3, C1, and C2 domains of porcine factor VIII (fVIII) have been cloned and sequenced. Along with previously determined sequences of the porcine fVIII B domain and the A2 domain, this completes the sequence determination of the cDNA corresponding to the translated protein. Alignments of the derived amino acid sequence of porcine fVIII with human and murine fVIII indicate that the A1, A2, A3, C1, and C2 domains are more conserved than the B domains or the proteolytic cleavage peptides corresponding to residues 337–372 and 1649–1689. The knowledge of the porcine fVIII cDNA may be useful to understand functional and immunological differences between human and porcine fVIII and may lead to improved fVIII replacement products for hemophilia. A patients through the development of recombinant porcine fVIII or hybrid human/porcine fVIII derivatives.

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