Light Chain Antigen Research Articles

Progress of chimeric antigen receptor T-cell therapy for multiple myeloma

Chimeric antigen receptor T-cell (CAR-T) therapy has made a breakthrough in the treatment of hematological tumors. CAR-T therapy targeting kappa immunoglobulin light chain, CD19 and B-cell mature antigen (BCMA) have been used to treat relapsed/refractory MM (RRMM) patients, and the effect of anti-BCMA CAR-T is the best. However, the negative or positive recurrence of BCMA leads to a short progression-free survival (PFS), the improvement of the chimeric antigen receptor (CAR) structure targeted BCMA, dual-targeted CAR-T and CAR-T combined with small molecular drugs as well as other measures have become the focuses of CAR-T in the treatment of RRMM. Key words: Multiple myeloma; Chimeric antigen receptor T-cell; B-cell maturation antigen; CD19

677. Stabilizing the rAAV Vector Genome Increases AAV Vector Transduction Efficiency

It has been determined that AAV vector efficiency decreases due to the following mechanisms: ineffective endocytosis, endosomal degradation, inefficient trafficking, and the need to convert from a single-stranded AAV genome to a transcriptional active double-stranded form. Administering rAAV vectors in conjunction with a drug that can enhance vector transduction may overcome some of these cellular barriers encountered during infection as well as help stabilize the genome. Here we discovered that Cidofovir(CDV), a monophosphate nucleotide analogue that competitively inhibits the incorporation of deoxycytidine triphosphate into viral DNA via viral DNA polymerase, is able to increase single-stranded rAAV transgene expression in the nucleus by 2 to 9 fold, depending on the cell type and concentration. This increase has been consistently observed in 16095, HelaS3, and Hek293 cells with little toxicity to the cells. However, there is less than a two-fold increase with Cidofovir treatment when using self-complementary AAV vectors(scAAV), suggesting that CDV might enhance conversion of the AAV genome from single to double stranded form. These observations were confirmed using two different serotypes, AAV2 and AAV8, in vitro indicating that the mechanism of enhancement is not serotype dependent. Southern Blot analysis along with real-time PCR demonstrated that Cidofovir treatment in HelaS3 cells increases viral DNA accumulation 24 hours post-infection compared to PBS treated cells. Cells pretreated with CDV then infected with rAAV revealed no difference in the amount of vector present between 0 and 2 hrs post infection, suggesting CDV does not enhance viral entry. However, cytoplasmic and nuclear fractions of HelaS3 cells pretreated with CDV or PBS demonstrated that CDV pretreatment enhances viral accumulation of rAAV vectors in both the cytoplasm and nucleus 24 hours post-infection.In vivo, Hemophilia A BALB/c(BALB/c-HA) mice were treated with CDV prior to infection with a rAAV containing the fVIII Human Light chain(LC) gene. It showed that CDV treatment enhanced the amount of LC antigen when compared to control group. Approximately two fold increase was observed up to fourteen weeks post-injection. These results demonstrate that CDV treatment can enhance and sustain rAAV levels in vivo.CDV has already been approved by the Food and Drug Administration (FDA) for the treatment of cytomegalovirus retinitis in patients with acquired immunodeficiency syndrome (AIDS). The effects of CDV on small DNA viruses that lack their own viral DNA polymerase, like AAV, have not been documented. The use of drugs like Cidofovir can greatly contribute to the understanding of rAAV trafficking and eventually lead the development of novel strategies to increase overall efficiency of AAV transduction.

The Analytical Performance Evaluation of Freelite™ Human Kappa Free and Human Lambda Free on the SPAPLUS™ Immunoturbidimetric Analyzer

SPAPLUS™ is a turbidimetric immunoassay analyzer for detection of excess free light chain (FLC) antigens in serum. Here, we evaluated the analytical performance of Freelite™ Human Kappa Free and Lambda Free on a SPAPLUS™ instrument. We evaluated the precision, linearity, sample carryover, and drift of the SPAPLUS™ instrument and compared it with Hitachi 7600 and BN™ II instruments. We evaluated the detection of antigen excess for 12 specimens from patients with monoclonal gammopathy. The coefficients of variations of κFLC and λFLC were below 5.0%. Linearity was shown in the range of 9.68-152.25 mg/l for κFLC and 4.96-171.09 mg/l for λFLC, and no drift was observed. The κFLC sample carryover was statistically significant, but much smaller than the optimum allowable bias. Agreement rates with the two comparative methods were 87.1, 87.1, and 97.1% or higher for κFLC, λFLC, and the κ/λ ratio, respectively. Antigen excess signals were observed for all 12 antigen excess specimens. The Freelite™ on the SPAPLUS™ exhibited appropriate precision, linearity, and relative comparability to the reagents on the other instruments. It was good at detecting specimens that had previously demonstrated the hook effect due to antigen excess.

Targeting of human CD34(+) hematopoietic stem cells with myosin light chain preserves cardiac function in chronic infarcted mouse heart

Background We demonstrated that transplantation of the engineered CD 34(+) hematopoietic stem cells [CD34(+)HSC] mitigates cardiac function loss in damaged mouse hearts. Objective: To determine 1) if targeted arming CD34(+)HSC with bivalent antibodies directed against myosin light chain (MLC) antigen increases its homing in myocardial infarction (MI); 2) if engineered CD34(+)HSC mitigates functional loss in mouse MI. Methods: MI was created by ligation of the left descending artery in adult mice. After 48 hr, animals were divided into 3 groups: 1) MI (n = 4): animals received RPMI medium; 2) MI + CD34(+)HSC (n = 7): animals received 5x105 CD34(+)HSC; 3) Sham (n = 4): animals underwent thoractomy without MI. Two weeks later, cardiac contractility was measured in Langendorff model. Immunohistochemistry (IHS) was used to monitor the homing of armed CD34+HSC. Results: MI heart exhibited enlarged chamber diameters and a decrease in contractility comparedwith sham hearts. IHS demonstrated an accumulation of CD34+HSC in MI hearts. Engraftment of CD34+HSC increased left ventricular developed pressure (Sham/MI/CD34(+)HSC = 95 ± 1/58 ± 12/99 ± 5 mmHg, *p<0.05), rate pressure product (Sham/MI/CD34(+)HSC = 44 ± 3 x10−3/24 ± 5 x10−3/43 ± 5 x10−3 mmHg.beats/min, *p<0.05), heart rate (Sham/MI/CD34(+)HSC = 475 ± 36 /405 ± 32/432 ± 33 beats/min). Conclusion: Targeted arming of CD34+HSC with MLC results in an increase in contractility in post MI heart. The protective effects of CD34(+)HSC in MI is associated with an increase in accumulation of engineered CD34+HSC in MI hearts. (NIH 1 P20 RR018728)

Characterization of an immunologic polymorphism (D79H) in the heavy chain of factor V

During the study of a family with hereditary factor (F)V deficiency (FV Amersfoort, 1102 A > T in exon 7) we identified an individual with 5% FV heavy chain antigen (FV(HC)) and 50% FV light chain antigen (FV(LC)). Further testing revealed that apart from the FV Amersfoort allele a second variant FV allele was segregating in this family, which encodes for a FV molecule with a reduced affinity for mAb V-23 used in the FV heavy chain ELISA (ELISA(HC)). Identification and characterization of the molecular basis responsible for the reduced affinity of the variant FV for mAb V-23. Family members of the proband were screened for mutations in the exons coding for the heavy chain of FV, after which the recombinant variant FV could be generated and characterized. Next, the cases and controls of the Leiden Thrombophilia Study (LETS) were genotyped for carriership of the variant FV. In the variant FV allele a polymorphism in exon 3 (409G > C) was identified, which predicts the replacement of aspartic acid 79 by histidin (D79H). Introduction of this mutation in recombinant FV confirmed that it reduces the affinity for binding to mAb V-23. The substitution has no effect on FV(a) stability and Xa-cofactor activity. In Caucasians the frequency of the FV-79H allele is approximately 5%. Analysis of the LETS revealed that the FV-79H allele is not associated with FV levels (FV(LC)), activated protein C sensitivity (using an activated partial thromboplastin time-based test) or risk of venous thrombosis (OR 1.07, CI 95: 0.7-1.7). The D79H substitution in FV should be considered as a neutral polymorphism. The monoclonal antibody V-23, which has a strongly reduced affinity for FV-79H, is not suitable for application in diagnostic tests.

Kinesin light chain in a eubacterium.

A eubacterial homolog of a kinesin light chain gene has been isolated and characterized from the cyanobacterium Plectonema boryanum. Although the eubacterial and eukaryotic kinesin light chains are highly similar in amino acid sequence, the eubacterial sequence differs in several distinguishing structural features, including the absence of a putative PEST domain and the presence of additional highly conserved imperfect tandem repeats. Two soluble kinesin light chain antigens have been identified from whole-cell lysates by immunoblot analysis. Attempts to identify a canonical kinesin heavy-chain gene or protein were unsuccessful, suggesting that a kinesin heavy chain may be absent or unnecessary for kinesin light-chain function in this eubacterium. Our findings establish that certain basal elements of eukaryotic cellular transport appear to be resident in eubacteria. We discuss the possibility that the eukaryotic kinesin light chain was acquired by lateral gene transfer.

Cofactors V and VIII after endotoxin administration to human volunteers

Coagulation factor V (FV) and factor VIII (FVIII) are usually decreased in septicemic DIC. Low doses of endotoxin administered to healthy volunteers stimulate activation of the fibrinolytic, contact and coagulation systems, but not clinical DIC. Following the administration of endotoxin (4 ng/kg) to normal volunteers (n = 15), we applied new assays for FV antigens using monoclonal antibodies to the activation peptide (C1) and to the light chain of FV. At 5 hours, FV coagulant activity was significantly decreased (64 ± 9%), as was the FV light chain antigen (74 ± 6%), without a change in factor V C1 antigen or total protein C. In contrast, FVIII coagulant activity was greater than preinfusion levels at 2–5 hours. The decrease in FV activity may be due to APC cleavage of FV heavy chain, but the loss of light chain antigen suggests that plasmin and/or calpain also contribute. APC may not be the only enzyme responsible for cofactor inactivation. FV is one of the most sensitive markers, even reflecting subclinical activation of coagulation.

Orbital Lymphoma Associated with Acquired Immune Deficiency Syndrome (AIDS)

A new case of AIDS-associated, small noncleaved cell (Burkitt's) lymphoma that presented as an orbital mass is described. Extraocular muscle involvement was documented by computed tomography and confirmed by orbital biopsy. Extensive abdominal involvement was subsequently diagnosed and this caused the patient's death only 15 days after the initial consultation and orbital biopsy. A literature review discloses only eight previous case reports of AIDS-associated non-Hodgkin's lymphoma (NHL) involving the orbit: two cases of Burkitt's type and six cases of large cell immunoblastic type. Three additional cases of orbital NHL can be found among larger series of AIDS cases. Affected patients are uniformly young adult males with a history of homosexuality and/or IV drug abuse. Variations in clinical presentation are discussed. Although the morphology of the histopathologic specimen in our case was consistent with a high-grade, small noncleaved cell NHL, flow cytometry revealed an atypical immunophenotype for Burkitt's lymphoma in that CD20 and immunoglobulin light chain antigens were not expressed. Morphologic classification and immunophenotypic characteristics of AIDS-associated NHLs are discussed.

Measurement of the Intensity of Cell Surface Antigen Expression in B-Cell Chronic Lymphocytic Leukemia

Immunophenotyping of peripheral blood lymphocytes from 95 patients with B-cell chronic lymphocytic leukemia (B-CLL) was performed to estimate surface membrane intensity of the HLA-DR, CD20, and light-chain antigens. Cases were classified as having weak, moderate, or strong intensity by a standard slide immunocytochemical method, and the fluorescence intensity (FI) was measured by flow cytometry in molecules of equivalent soluble fluorescence (MESF). Staining intensity was strongest for HLA-DR; with both methods, 94 cases were classified as strong. For CD20, 67% were weak, 27% moderate, and 6% strong; the mean MESF values were 31.7, 66.4, and 177.4 x 10(3), respectively (P < .001). Thirty-nine percent had weak surface light-chain staining, 50% moderate, and 11% strong; the mean MESF values were 4.9, 7.5, and 16 x 10(3), respectively (P = .005). These results indicate that the FI of cell surface antigens can be estimated and quantified by flow cytometry. Because of the wide overlap of FI values among the three groups, an exact correlation was not found between the two methods of classifying intensity of surface antigen expression. The usefulness of these quantitative measurements needs further analysis in clinical trials.

モノクローナル抗体カラム法で製造された高純度第ＶＩＩＩ因子製剤（クロスエイトＭ）中の第ＶＩＩＩ因子の生化学的性質

Biochemical properties of Factor VIII (FVIII) in highly-purified FVIII concentrates (CROSS EIGHT M) produced by monoclonal antibody column were studied and compared with those of Hemofil M which was already used clinically. The FVIII clotting activity (FVIII:Cclot) levels obtained by one-stage method using severe hemophilia A plasma as a substrate were 614-729U/vial and identical with those indicated on the label (500U/vial). The FVIII light chain antigen (FVIIIL:Ag) levels measured by Enzyme Immunoassay were 540-760U/vial. The ratio of FVIIIL:Ag to FVIII:Cclot (FVIIIL:Ag/FVIII:Cclot) were 0.88-1.04. The ratio of FVIIIL:Ag to von Willebrand Factor antigen (vWF:Ag) (FVIIIL:Ag/vWF:Ag) were 49.0-60.8. No difference was observed between CROSS EIGHT M and Hemofil M on the FVIII:Cclot levels, FVIIIL:Ag levels, FVIIIL:Ag/FVIII:Cclot and FVIIIL:Ag/vWF:Ag.SDS-PAGE patterns showed almost no contamination except albumin being added as a stabilizer. Western blot analysis revealed FVIII heavy chain antigen of M. W. 90kDa-220kDa and FVIII light chain antigen of M. W. 80kDa. No degradates of MW. 43kDa-50kDa derived from FVIII heavy chain antigen were observed. CROSS EIGHT M lacked the large multimers of vWF which were detected in normal human pooled plasma. No difference was observed between CROSS EIGHT M and Hemofil M by western blot analysis of FVIII and multimer analysis of vWF.These results suggest that CROSS EIGHT M preserves the fundamental structure of FVIII molecule for coagulant activity. Therefore, CROSS EIGHT M as well as Hemofil M may be clinically used with safety as FVIII concentrates.

Single-chain site-specific mutations of fluorescein-amino acid contact residues in high affinity monoclonal antibody 4-4-20

Previous crystallographic studies of high affinity anti-fluorescein monoclonal antibody 4-4-20 (Ka = 1.7 x 10(10) M-1) complexed with fluorescyl ligand resolved active site contact residues involved in binding. For better definition of the relative roles of three light chain antigen contact residues (L27dhis, L32tyr and L34arg), four site-specific mutations (L27dhis to L27lys, L32tyr to L32phe, and L34arg to L34lys and L34his) were generated and expressed in single-chain antigen binding derivatives of monoclonal antibody 4-4-20 containing two different polypeptide linkers (SCA 4-4-20/205c, 25 amino acids and SCA 4-4-20/212, 14 amino acids). Results showed that L27dhis and L32tyr were necessary for wild type binding affinities, however, were not required for near-wild type Qmax values (where Qmax is the maximum fluoroscein fluorescence quenching expressed as percent). Tyrosine L32 which hydrogen bonds with ligand was also characterized at the haptenic level through the use of 9-hydroxyphenylfluoron which lacks the carboxyl group to which L32 tyrosine forms a hydrogen bond. Results demonstrated that wild type SCA and mutant L32phe possessed similar HPF binding characteristics. Active site contact residue L34arg was important for fluorescein quenching maxima and binding affinity (L34his mutant), however, substitution of lysine for arginine at L34 did not have a significant effect on observed Qmax value. In addition, substitutions had no effect on structural and topological characteristics, since all mutants retained similar idiotypic and metatypic properties. Finally, two linkers were comparatively examined to determine relative contributions to mutant binding properties and stability. No linker effects were observed. Collectively, these results verified the importance of these light chain fluorescein contact residues in the binding pocket of monoclonal antibody 4-4-20.

An immunohistochemical study of spontaneous lymphomas in the C57B1/10J mouse

Immunocytochemistry was used to examine 26 cases of composite lymphoma in the mouse mesenteric lymph node. The diagnosis was made by light microscopic criteria. A selection of polyclonal antibodies to light (Kappa and Lambda) and heavy chain (IgD, IgM, IgC, IgA, IgE) antigens was used together with three monoclonal antibodies to T cells, B cells (HLA-DR) and macrophages. Twenty lymphomas were classified as B cell and six as T cell. The B cells were subdivided into IgM and IgD (five), IgD (five), IgM (three) and IgA (one), three undifferentiated and, in three, there was insufficient tissue for further typing. There did not appear to be any correlation between the morphological appearance and the immunophenotype.

Immunocytochemical identification and localization of immunoglobulin A within Paneth cells of the rat small intestine.

Light microscopic immunocytochemistry was used to identify Paneth cells by their lysozyme content and to detect immunoglobulin antigens within a subpopulation of these cells. Antisera specific for the heavy chains of rat or human immunoglobulin A and for immunoglobulin light chain antigens produced specific staining of rat Paneth cells. The distribution of immunoglobulin staining varied between adjacent Paneth cells in the same crypt and between Paneth cells in adjacent crypts, as well as between Paneth cell populations of different animals. No staining of rat Paneth cells was detected using antisera specific for the heavy chain of immunoglobulins G or M. The specific staining of Paneth cells for immunoglobulin A and light chain antigens was blocked by absorption of each antiserum with its respective purified antigen. Absorption of these antisera with purified rat lysozyme did not affect staining and thereby eliminated the possibility of immunologic cross-reactivity between lysozyme and immunoglobulin antigens. It is suggested, in light of current concepts of Paneth cell function, that the immunoglobulin staining of Paneth cells may reflect their ability to phagocytize immunoglobulin A-coated microorganisms or immune complexes containing immunoglobulin A.

Immunochemical studies on factor V.

Native bovine factor V exhibits a molecular weight of 300000 as determined by gel filtration of untreated plasma. Highly purified factor V exhibits multiple molecular weight forms which range from small active fragments to aggregates of several million which are generated during the purification on cellulose phosphate. Isoelectric focusing on a single high-molecular-weight species produced a single protein and activity peak at pH 4.65. Factor V activity is associated with each protein band observed following polyacrylamide gel electrophoresis. Antisera to factor V prepared in rabbits produces a time-dependent and concentration-dependent inhibition of factor V activity in plasma and purified factor V. The multiple molecular weight forms of factor V appear equivalent upon immunodiffusion and on immunoelectrophoresis migrate as an alpha globulin between albumin and fibrinogen. Immunoprecipitation arcs are equivalent in plasma and serum. Factor V consists of two major types of subunits, a light chain (73000), aggregates of which form the high-molecular-weight species, and a heavy chain (125 000). Using preparations containing one or both chains isolated by disc gel electrophoresis, antiserum was shown to contain two families of antibodies, one against each subunit. Cross reactivity with both light and heavy chain antigens is observed in sheep and goat but not monkey or human plasma. The antisera also neutralized goat and sheep factor V activity.

Maternal antibody to fetal light chain (Inv) antigens

We report a case of immunization by fetal light (L) chain (Inv) antigens in a primipara who had never received a transfusion. An Inv(l−a−) mother produced an immunoglobulin M anti-Inv(l) antibody in high titer to her Inv(l+a+) child. The child had an abnormal placenta and died of neonatal nephrosis at 6 months. A second child was born 3 years later. This infant's L chains were also Inv(l+a+), but the mother demonstrated no immunologic recognition of the incompatible Inv antigen as demonstrated by her anti-Inv(l) titer. The second child and his placenta were normal, and at 10 months of age he was found to have an anti-Gm(a) antibody in high titer to a maternal IgG H chain antigen. Anti-Gm antibodies in mothers and children are not uncommon and are usually not associated with noticeable disease, but the pathophysiology of maternal anti-lnv antibodies is not known.

Evolution of the immunoglobulin antigens in the ruminantia.

The cross-reactivity of the immunoglobulin γ, γ 1 , γ 2 , α, μ, and light chain antigens of 21 ruminant species was examined using an inhibition of radioimmune precipitation technique. Although it was clear that the different antigens had evolved at different rates, the relative ranking of cross-reactivity correlated well with the accepted taxonomic order of the ruminants. It was considered significant that Antilocapra was closer to the bovids than to the cervids, in view of the disputed relationship of that genus to these families. Further, it was noted that sheep and goats appeared to be no more or less closely related than, say, the cattle and the antelopes. Of the IgG subclass antigens, IgG 1 showed the higher evolutionary stability, strong cross-reactivity being observed between sheep and deer and even between sheep and camel. This stability might be due to the conservation of sequences related to the biological properties of IgG 1 —its ability to fix complement and its secretion into the colostrum and absorption in the gut of the newborn.

Cultured lymphoid cell lines from normal subjects: Membrane associated immunoglobulins studied by the mixed antiglobulin reaction

The mixed antiglobulin reaction was used to analyze the membrane associated immunoglobulins on lymphoid cells of established suspension cultures grown from the peripheral blood of seven healthy individuals and three subjects with viral diseases. After preincubation in an acetate buffer system, membrane immunoglobulins were found on a high percentage of cells; in control samples not exposed to acetate buffer less than 4% of the cells were positive. All 10 lines had κ light chain antigens on 5 to 83% of cells; 8 had μ antigens on 28 to 91% of cells, two lines were μ negative. IgG Fc, IgA and IgD antigens were not detected. The immunological specificity of the detection systems was confirmed by inhibition with isolated light chains, F(ab′) 2 fragments, or intact molecules from purified monoclonal proteins of the various immunoglobulin classes and chain types. Cultured cells were synchronized by double thymidine blockade and examined. Except for a small decrease in the late G 2 phase, the proportion of lymphoid cells with membrane associated μ and κ antigens remained relatively constant throughout the cell cycle.

Alterations in Isotypic and Allotypic Immunoglobulin Antigens Induced by Chemical Modification

Abstract Gamma G myeloma and Bence Jones protein were modified by amidination, a treatment specific for ε-amino groups of lysine, and tested before and after modification for their serologic reactivity with antisera against defined γ and light chain antigens. Gm(a), “non-a”, Gm(z), Inv(a), and Oz(+) reactivity but not that of Gm(f), (b0), (g) or “non-g” were inactivated by this treatment. Lysine residues are known to be directly involved or lie immediately adjacent to the amino acid positions responsible for expression of all the antigens altered by amidination. It is suggested that the use of chemical probes specific for various amino acid resides may be of benefit in defining the structures responsible for the serologically-defined antigens of human immunoglobulins.

Demonstration of heavy and light chain antigenic determinants on the cell-bound receptor for antigen. Similarities between membrane-attached and humoral antibodies produced by the same cell.

High-rate antibody-forming cells and immunological memory cells can be selectively retained if filtered through a column coated with relevant antigen. This trapping can be blocked if the cells are incubated with an anti-immunoglobulin serum prior to column passage. A similar blocking is not observed when cells are treated with an anti-lymphocyte serum, thereby excluding the possibility that any antibodies combining with surface structures could cause this effect. By the use of antisera specific for heavy or light chain antigens, it was possible to locate such antigens in the antigen-binding receptor areas of immune cells. Criss-cross studies using antisera specific for gamma 1 or gamma 2a heavy chains showed that the membrane receptor has the same heavy chain as will be present in the eventual product of that cell, the humoral antibody.

Biosynthetic and structural studies of a heavy chain disease protein.

A new heavy chain disease protein ((gamma)HCD-JM) has been characterized by antigenic and structural criteria. The protein belongs to the IgG3-subclass and is closely related to Fc-fragment of G3-immunoglobulins. The predominant N-terminal amino acid of this protein is glutamic acid in the uncyclized form, and that of another (gamma)HCD is glycine. Studies of the N-terminal peptides indicate that the N-terminal portion of the (gamma)3-heavy polypeptide chain is absent from the (gamma)HCD-JM. These findings rule out a process of normal heavy chain initiation and a large deletion of the Fd region as being responsible for these two heavy chain disease proteins. The (gamma)HCD-JM is a secretory product of cells from bone marrow as shown by studies of in vitro incorporation of amino acids-(14)C. Bone marrow and lymph node have a population of lymphoplasmacytic cells which by immunofluorescence contain (gamma)-heavy chain antigens in the absence of light chain antigens.