Therapeutic Potential of HLA-I Polyreactive mAbs Mimicking the HLA-I Polyreactivity and Immunoregulatory Functions of IVIg

HLA class-I (HLA-I) polyreactive monoclonal antibodies (mAbs) reacting to all HLA-I alleles were developed by immunizing HLA-E monomeric heavy chain (HC) (Open Conformers, OCs). Two of the mAbs (TFL-006 and TFL-007) bound to the HC’s coated on a solid matrix. The binding was inhibited by a peptide 117AYDGKDY123, present in all alleles of the six HLA-I isoforms but masked by 2-microglobulin -m) in intact HLA-I trimers (Closed Conformers, CCs). Identical HLA-I polyreactivity is observed in IVIg administered to lower anti-HLA antibodies (Abs) in HLA-sensitized patients, but the mechanism is unknown. We hypothesized that the mAbs that mimic IVIg HLA-I polyreactivity might mimic the immunomodulatory functions of IVIg. We tested the relative binding affinity of the mAbs and IVIg for both OCs- and CCs and compared their effects on (a) the phytohemagglutinin (PHA)-activation T-cells, (b) the production of anti-HLA-II antibody (Ab) by B-memory cells, and anti-HLA-I Ab by immortalized B-cells, and (c) the upregulation of CD4+, CD25+, and Fox P3+ T-regs. The mAbs bound only to OCs, whereas IVIg is bound to both CCs and OCs. The mAbs suppressed blastogenesis and proliferation of PHA-activated T-cells, anti-HLA Ab production by B-cells and expanded the T-regs, better than IVIg. We conclude that a humanized version of the TFL-mAbs could be an ideal therapeutic IVIg-mimetic.

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Therapeutic Potential of HLA-I Polyreactive mAbs Mimicking the HLA-I Polyreactivity and Immunoregulatory Functions of IVIg

Vaccines ◽

10.3390/vaccines9060680 ◽

2021 ◽

Vol 9 (6) ◽

pp. 680

Author(s):

Mepur H. Ravindranath ◽

Fatiha El Hilali ◽

Edward J. Filippone

Keyword(s):

T Cells ◽

B Cells ◽

Therapeutic Potential ◽

Hla Class I ◽

Solid Matrix ◽

Binding Affinities ◽

Activated T Cells ◽

Transplant Patients ◽

Relative Binding ◽

Better Than

HLA class-I (HLA-I) polyreactive monoclonal antibodies (mAbs) reacting to all HLA-I alleles were developed by immunizing mice with HLA-E monomeric, α-heavy chain (αHC) open conformers (OCs). Two mAbs (TFL-006 and TFL-007) were bound to the αHC’s coated on a solid matrix. The binding was inhibited by the peptide 117AYDGKDY123, present in all alleles of the six HLA-I isoforms but masked by β2-microglobulin (β2-m) in intact HLA-I trimers (closed conformers, CCs). IVIg preparations administered to lower anti-HLA Abs in pre-and post-transplant patients have also shown HLA-I polyreactivity. We hypothesized that the mAbs that mimic IVIg HLA-I polyreactivity might also possess the immunomodulatory capabilities of IVIg. We tested the relative binding affinities of the mAbs and IVIg for both OCs and CCs and compared their effects on (a) the phytohemagglutinin (PHA)-activation T-cells; (b) the production of anti-HLA-II antibody (Ab) by B-memory cells and anti-HLA-I Ab by immortalized B-cells; and (c) the upregulation of CD4+, CD25+, and Fox P3+ T-regs. The mAbs bound only to OC, whereas IVIg bound to both CC and OC. The mAbs suppressed blastogenesis and proliferation of PHA-activated T-cells and anti-HLA Ab production by B-cells and expanded T-regs better than IVIg. We conclude that a humanized version of the TFL-mAbs could be an ideal, therapeutic IVIg-mimetic.

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THU0036 FIRST-IN-HUMAN TRIAL OF BCMA-CD19 COMPOUND CAR IN THE TREATMENT OF AUTOANTIBODY MEDIATED DISORDERS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.4065 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 231.1-231

Author(s):

F. Liu ◽

H. Zhang ◽

X. Wang ◽

J. Feng ◽

Y. Cao ◽

...

Keyword(s):

Clinical Trial ◽

T Cells ◽

B Cells ◽

Plasma Cells ◽

Phase 1 ◽

Human Clinical Trial ◽

Hla Antibodies ◽

Time Points ◽

Car T ◽

Reh Cells

Background:Donor-specific anti-HLA antibodies (DSAs) are antibodies in the recipient directed against donor class I/II HLA antigens. The existence of DSAs before allogenic hematopoietic stem cell transplantation (AHSCT) are known to cause primary graft failure. Currently there’s no established method of DSA desensitization due to the long half-life of plasma cells.Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease involving in multiple organ systems mediated by numerous autoantibodies. Recent results have shown that depletion of B cells by CD19 CAR-T cells effectively reversed some manifestations in two SLE mouse models. However, plasma cells could be spared with single CD19 CAR-T cells, and peripheral circulating anti-DNA IgG and IgM autoantibodies remain elevated or increased in treated mice.Objectives:We present the efficacy of BCMA-CD19 compound CAR (cCAR), which target on antibody- producing “root”, both B cells and plasma cells in preclinical study and in our first-in-human phase 1 clinical trial.Methods:We constructed a BCMA-CD19 cCAR composed of a complete BCMA-CAR fused to a complete CD19 CAR, separated by a self-cleaving P2A peptide. We assessed the functional activity of cCAR in co-culture assay with multiple cell lines. We also verified cCAR efficacy with two mouse models, injected with either BCMA-expressing MM.1S cells or CD19-expressing REH cells. In our phase 1 clinical trial, we enrolled patients with hematologic malignancies with antibody mediated disorders.Results:BCMA-CD19 cCAR exhibited robust cytotoxic activity against the K562 cells engineered to express either CD19 or BCMA in co-culture assays, indicating the ability of each complete CAR domain to specifically lyse target cells. In mouse model study, cCAR-T cells were able to eliminate tumor cells in mice injected with MM.1S cells and REH cells, indicating that both BCMA and CD19 are specifically and equally lysing B cells and plasma cells in vivo, making BCMA-CD19 cCAR a candidate for clinical use.In our first-in-human clinical trial, the first case is a 48-year-old female patient having resistant B-ALL with high DSA titers. She exhibited complete remission of B-ALL at day 14 post-CAR T treatment. MFI of DSA dropped from 7800 to 1400 at 8 weeks post cCAR treatment, the reduction percentage was approximately 80% (Figure 1). The patient had no CRS, and no neurotoxicity was observed.Figure 1.1. A) MFI of DSA and other HLA antibodies before and at different time points after cCAR T infusion. B) the percent reduction post-transfusion of cCAR T cells at different time points.The second case is a 41-year-old female patient having a refractory diffuse large B cell lymphoma with bone marrow (BM) involvement. Furthermore, she has a 20 years of SLE, with manifestation of fever dependent of corticosteroids. On day 28 after cCAR treatment, PET/CT scan showed CR, and BM turned negative. In addition, she is independent of steroids, has no fever and other manifestations, C3/C4 are within normal ranges, and all the ANA dropped significantly, especially the nuclear type ANA, which turned from> 1:1000 to be negative at day 64. She had Grade 1 CRS but with no neurotoxicity observed. The absence of B cells and plasma cells persisted more than 5 months post CAR therapy.Conclusion:Our first in human clinical trial on BCMA-CD19 cCAR demonstrated profound efficacy in reducing DSA levels in an AHSCT candidate and ANA titer in a SLE patient. There was strong clinical evidence of depletion of antibody-producing roots, B-cells and plasma cells in both patients. Our results further suggested that BCMA-CD19 cCAR has the potential to benefit patients receiving solid organ transplants or those with other antibody-mediated diseases.Figure 2.Reduction of different type of ANA titer at different time points.Acknowledgments:patients and their familiesDisclosure of Interests:Fang liu: None declared, Hongyu Zhang: None declared, Xiao Wang: None declared, Jia Feng: None declared, Yuanzhen cao Employee of: Employee of iCell Gene Therapeutics LLC, Yi Su: None declared, Masayuki Wada Employee of: employee of iCell Gene Therapeutics LLC, Yu Ma Employee of: employee of iCAR Bio Therapeutics Ltd, Yupo Ma Shareholder of: shareholder of iCell Gene Therapeutics LLC

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Short-Term Immunopathological Changes Associated with Pulse Steroids/IVIG/Rituximab Therapy in Late Kidney Allograft Antibody Mediated Rejection

Kidney360 ◽

10.34067/kid.0001082019 ◽

2020 ◽

Vol 1 (5) ◽

pp. 389-398

Author(s):

Kenna R. Degner ◽

Nancy A. Wilson ◽

Shannon R. Reese ◽

Sandesh Parajuli ◽

Fahad Aziz ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Regulatory T Cells ◽

B Cell ◽

Cd8 T Cells ◽

Hla Class I ◽

B Cell Depletion ◽

Short Term ◽

Antibody Mediated Rejection ◽

Pulse Steroids

BackgroundB cell depletion is a common treatment of antibody-mediated rejection (ABMR). We sought to determine the specific immunopathologic effects of this therapeutic approach in kidney transplantation.MethodsThis was a prospective observational study of recipients of kidney transplants diagnosed with late ABMR (>3 months after transplant). Patients received treatment with pulse steroids, IVIG, and rituximab. Donor-specific HLA antibodies (DSA), kidney allograft pathology, renal function, immune cell phenotypes, and 47 circulating cytokines were assessed at baseline and at 3 months.ResultsWe enrolled 23 patients in this study between April 2015 and March 2019. The majority of patients were male (74%) and white (78%) with an average age of 45.6±13.8 years. ABMR was diagnosed at 6.8±5.9 years (4 months to 25 years) post-transplant. Treatment was associated with a significant decline in circulating HLA class I (P=0.003) and class II DSA (P=0.002) and peritubular capillaritis (ptc; P=0.04) compared to baseline. Serum creatinine, BUN, eGFR, and proteinuria (UPC) remained stable. Circulating B cells were depleted to barely detectable levels (P≤0.001), whereas BAFF (P=0.0001), APRIL (P<0.001), and IL-10 (P=0.02) levels increased significantly post-treatment. Notably, there was a significant rise in circulating CD4+ (P=0.02) and CD8+ T cells (P=0.003). We also noted a significant correlation between circulating cytotoxic CD8+ T cells and BAFF (P=0.05), regulatory T cells and IL-10 (P=0.002), and regulatory T cells and HLA class I DSA (P=0.005).ConclusionsShort-term pulse steroids/IVIG/rituximab therapy was associated with inhibition of ABMR (DSA and ptc), stabilization of kidney function, and increased regulatory B cell and T cell survival cytokines. Additional studies are needed to understand the implications of B cell depletion on the crosstalk between T cells and B cells, and humoral components that regulate ABMR.

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IMMUNOLOGIC REACTIONS TO HAPTENS ON AUTOLOGOUS CARRIERS

Journal of Experimental Medicine ◽

10.1084/jem.137.2.411 ◽

1973 ◽

Vol 137 (2) ◽

pp. 411-423 ◽

Cited By ~ 27

Author(s):

John W. Moorhead ◽

Curla S. Walters ◽

Henry N. Claman

Keyword(s):

T Cells ◽

B Cells ◽

Aminocaproic Acid ◽

Single Amino Acid ◽

Secondary Response ◽

Spleen Cells ◽

Activated T Cells ◽

Thymus Cells

Both thymus-derived (T) and bone marrow-derived (B) lymphocytes participate in the response to a hapten 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP), coupled to a nonimmunogenic isologous carrier, mouse gamma globulin (MGG). Spleen cells from mice immunized with NIP-MGG show increased DNA synthesis in vitro when cultured with NIP-MGG. The participation of and requirement for T cells in the response was demonstrated by treating the spleen cells with anti-θ serum. This treatment resulted in a 77% inhibition of the antigen response. Furthermore, adoptively transferred normal thymus cells could be specifically "activated" by NIP-MGG in vivo and they responded secondarily to the antigen in vitro. The active participation of B cells in the secondary response was demonstrated by passing the immune spleen cells through a column coated with polyvalent anti-MGG serum. Column filtration reduced the number of NIP-specific plaque-forming cells and NIP-specific rosette-forming cells (both functions of B cells) and produced a 47% inhibition of the NIP-MGG response. The ability of the cells to respond to phytohemagglutinin (PHA) was not affected by column filtration showing that T cells were not being selectively removed. The participation of B cells in the in vitro NIP-MGG response was also shown by treatment of the spleen cells with antiserum specific for MGG and MGG determinants. B cells were removed by treatment with anti-IgM or polyvalent anti-MGG serum plus complement, resulting in a respective 46 and 49% inhibition of the response to NIP-MGG. (Treatment with anti-IgM serum had no effect on T cells.) The contribution of the hapten NIP to stimulation of T cells was investigated using NIP-MGG-activated thymus cells. These activated T cells responded in vitro very well to the NIP-MGG complex but not to the MGG carrier alone demonstrating the requirement of the hapten for T cell stimulation. The response was also partially inhibited (41%) by incubating the activated cells with NIP coupled to a single amino acid (epsilon-aminocaproic acid) before addition of NIP-MGG. These results demonstrated that T cells recognize the hapten NIP when it is coupled to the isologous carrier MGG.

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Lack of Sprouty 1 and 2 enhances survival of effector CD8+ T cells and yields more protective memory cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1808320115 ◽

2018 ◽

Vol 115 (38) ◽

pp. E8939-E8947 ◽

Cited By ~ 8

Author(s):

Hesham M. Shehata ◽

Shahzada Khan ◽

Elise Chen ◽

Patrick E. Fields ◽

Richard A. Flavell ◽

...

Keyword(s):

T Cells ◽

Memory T Cells ◽

Therapeutic Potential ◽

Autoimmune Diabetes ◽

Memory Cells ◽

Double Knockout ◽

And Function ◽

I Cells ◽

Better Than

Identifying novel pathways that promote robust function and longevity of cytotoxic T cells has promising potential for immunotherapeutic strategies to combat cancer and chronic infections. We show that sprouty 1 and 2 (Spry1/2) molecules regulate the survival and function of memory CD8+ T cells. Spry1/2 double-knockout (DKO) ovalbumin (OVA)-specific CD8+ T cells (OT-I cells) mounted more vigorous autoimmune diabetes than WT OT-I cells when transferred to mice expressing OVA in their pancreatic β-islets. To determine the consequence of Spry1/2 deletion on effector and memory CD8+ T cell development and function, we used systemic infection with lymphocytic choriomeningitis virus (LCMV) Armstrong. Spry1/2 DKO LCMV gp33-specific P14 CD8+ T cells survive contraction better than WT cells and generate significantly more polyfunctional memory T cells. The larger number of Spry1/2 DKO memory T cells displayed enhanced infiltration into infected tissue, demonstrating that absence of Spry1/2 can result in increased recall capacity. Upon adoptive transfer into naive hosts, Spry1/2 DKO memory T cells controlled Listeria monocytogenes infection better than WT cells. The enhanced formation of more functional Spry1/2 DKO memory T cells was associated with significantly reduced mTORC1 activity and glucose uptake. Reduced p-AKT, p-FoxO1/3a, and T-bet expression was also consistent with enhanced survival and memory accrual. Collectively, loss of Spry1/2 enhances the survival of effector CD8+ T cells and results in the formation of more protective memory cells. Deleting Spry1/2 in antigen-specific CD8+ T cells may have therapeutic potential for enhancing the survival and functionality of effector and memory CD8+ T cells in vivo.

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Production and characterization of a bispecific IgG capable of inducing T-cell-mediated lysis of malignant B cells

Blood ◽

10.1182/blood.v81.12.3343.3343 ◽

1993 ◽

Vol 81 (12) ◽

pp. 3343-3349 ◽

Cited By ~ 10

Author(s):

BK Link ◽

GJ Weiner

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Hybridoma Cells ◽

Activated T Cells ◽

Human B Cells ◽

B Cell Malignancy ◽

Cell Malignancy

Abstract Bispecific monoclonal antibodies (bsabs) recognizing both CD3 and a tumor antigen can redirect T-cell-mediated cytotoxicity toward cells bearing that antigen. Such bsabs have been shown to be more effective than monospecific monoclonal antibodies (MoAbs) at preventing tumor growth in animal models of B-cell malignancy. The current studies describe the production and preliminary evaluation of a bsab designed to induce the lysis of malignant human B cells by human T cells. The bsab was obtained from a hybrid-hybridoma cell line produced by fusing OKT3-secreting hybridoma cells with hybridoma cells that secrete 1D10. 1D10 is an MoAb that recognizes an antigen found on a majority of malignant human B cells that has not been detected to a significant degree on normal resting or activated lymphocytes. High performance liquid chromatography (HPLC) was used to separate bsab from monospecific antibodies that were also present in the hybrid-hybridoma antibody product. The bsab was then evaluated in vitro for its ability to induce lysis of malignant B cells by activated T cells. The bsab consistently induced extensive lysis in vitro of 1D10 (+) cells, including both cell lines and cells obtained from patients with a variety of B-cell malignancies. No such effect was seen with activated T cells alone or activated T cells with monospecific antibody. No increased lysis was seen with 1D10 (-) cell lines. The bsab also mediated lysis of malignant B cells by autologous T cells. We conclude bsab containing an OKT3 arm and a 1D10 arm can induce T-cell-mediated lysis in a manner that is both potent and specific. This supports further evaluation of this bsab as a potential immunotherapy of B-cell malignancy.

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Fas-Independent Apoptosis of Activated T Cells Induced by Antibodies to the HLA Class I α1 Domain

Blood ◽

10.1182/blood.v90.9.3629 ◽

1997 ◽

Vol 90 (9) ◽

pp. 3629-3639 ◽

Cited By ~ 32

Author(s):

Laurent Genestier ◽

Romain Paillot ◽

Nathalie Bonnefoy-Berard ◽

Geneviéve Meffre ◽

Monique Flacher ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Heavy Chain ◽

Hla Class I ◽

Class I ◽

T Cell Proliferation ◽

Activated T Cells ◽

Hla Class I Molecules

Abstract In addition to their major function in antigen presentation and natural killer cell activity regulation, HLA class I molecules may modulate T-cell activation and proliferation. Monoclonal antibodies (MoAbs) that recognize distinct epitopes of HLA class I molecules were reported to interfere with T-cell proliferation. We show here that two MoAbs (mouse MoAb90 and rat YTH862) that bind to an epitope of the α1 domain of HLA class I heavy chain induce apoptotic cell death of activated, but not resting, peripheral T lymphocytes. Other reference anti-HLA class I antibodies specific for distinct epitopes of the α1 (B9.12.1), α2 (W6/32), or α3 (TP25.99) domains of the heavy chain decreased T-cell proliferation but had little or no apoptotic effect. Apoptosis shown by DNA fragmentation, phosphatidylserine externalization, and decrease of mitochondrial transmembrane potential was observed whatever the type of T-cell activator. Apoptosis did not result from Fas/Fas-L interaction and distinct though partly overlapping populations of activated T cells were susceptible to Fas– and HLA class I–mediated apoptosis, respectively. Induction of apoptosis did not require HLA class I cross-linking inasmuch as it could be observed with monovalent Fab′ fragments. The data indicate that MoAb90 and YTH862 directed against the α1 domain of HLA class I trigger apoptosis of activated T lymphocytes by a pathway which does not involve Fas-ligand.

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Selective inhibition of growth factor-dependent human B cell proliferation by monoclonal antibody AB1 to an antigen expressed by activated B cells.

Journal of Experimental Medicine ◽

10.1084/jem.160.6.1919 ◽

1984 ◽

Vol 160 (6) ◽

pp. 1919-1924 ◽

Cited By ~ 11

Author(s):

L K Jung ◽

S M Fu

Keyword(s):

Monoclonal Antibody ◽

Cell Proliferation ◽

T Cells ◽

B Cells ◽

B Cell ◽

Leukemic Cells ◽

Activated T Cells ◽

Igm Antibodies ◽

Stimulatory Factor ◽

Activated B Cells

A monoclonal antibody, AB1, was established with activated human B cells as immunogen. AB1 stained activated B cells but not activated T cells. Its selective reactivity to activated B cells was further documented by its nonreactivity to activated T cells, resting T and B cells, monocytes, granulocytes, bone marrow cells, leukemic cells, and cells from cell lines of T, B, and myeloid lineages. Upon activation, the antigen appeared on B cells as early as 3-4 h after stimulation and was fully expressed by 38 h. The expression of this antigen was not dependent on the presence of B cell stimulatory factor(s). Anti-IgM antibodies by themselves induced its expression. AB1 inhibited B cell proliferation that was induced by a low dose anti-IgM antibody and conditioned medium containing B cell stimulatory factor. It did not inhibit B cell proliferation induced by either high doses of anti-IgM antibodies or by formalinized Staphylococcus aureus. It also failed to inhibit T cell mitogenesis. The possibility exists that this antigen is related to the receptor for B cell stimulatory factor.

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