InVivoMAb anti-human CD2

in vitro inhibition of MLR

Sellberg F, Berglund D, Binder C, Hope J, Fontenot J, Griesemer A, Sykes M, Sachs DH, Berglund E. (2020). "Pharmacokinetic and pharmacodynamic study of a clinically effective anti-CD2 monoclonal antibody" Scand J Immunol 91(1):e12839. PubMed

The humanized IgG1κ monoclonal antibody siplizumab and its rat parent monoclonal IgG2b antibody BTI-322 are directed against the CD2 antigen. Siplizumab is species-specific, reacting with human and chimpanzee cells but not with cells from any other species, including other non-human primates. Because siplizumab treatment has recently shown great potential in clinical transplantation, we now present the results of our previous pharmacokinetic, pharmacodynamic and safety studies of both antibodies. Fourteen chimpanzees received 1-3 doses of 0.143 to 5.0 mg/kg iv The effects were followed with flow cytometry on peripheral lymphocytes and staining of lymph nodes. Side effects were recorded. Serum antibody concentrations were followed. Across the doses, a rapid, transient depletion of CD2, CD3, CD4 and CD8 lymphocytes and NK cells was observed for both antibodies. Immune reconstitution was more rapid for BTI-322 compared to siplizumab. Paracortical lymph node T cell depletion was moderate, estimated at 45% with doses of >0.6 mg/kg. Restoration of lymph node architecture was seen after two weeks to two months for all animals. All four subjects receiving BTI-322 experienced AEs on the first dosing day, while the eight subjects dosed with siplizumab experienced few mild, transient AEs. Infusion with siplizumab and BTI-322 resulted in rapid depletion of CD2⁺ cells in circulation and tissue. Siplizumab had a longer t_1/2 and fewer AEs compared to BTI-322.

in vivo T cell depletion, in vivo prevention of graft rejection

Habiro K, Sykes M, Yang YG. (2009). "Induction of human T-cell tolerance to pig xenoantigens via thymus transplantation in mice with an established human immune system" Am J Transplant 9(6):1324-9. PubMed

Thymus xenotransplantation has been shown to induce tolerance to porcine xenografts in mice and to permit survival of alpha1,3Gal-transferase knockout porcine kidney xenografts for months in nonhuman primates. We evaluated the ability of porcine thymus xenotransplantation to induce human T-cell tolerance using a humanized mouse (hu-mouse) model, where a human immune system is preestablished by implantation of fetal human thymus tissue under the kidney capsule and intravenous injection of CD34(+) hematopoietic stem/progenitor cells. Human T-cell depletion with an anti-CD2 mAb following surgical removal of human thymic grafts prevented the initial rejection of porcine thymic xenografts in hu-mice. In these hu-mice, porcine thymic grafts were capable of supporting human thymopoiesis and T-cell development, and inducing human T-cell tolerance to porcine xenoantigens. Human T cells from these mice responded strongly to third-party pig, but not to the thymic donor swine leukocyte antigen (SLA)-matched pig stimulators in a mixed lymphocyte reaction (MLR) assay. Anti-pig xenoreactive antibodies declined in these hu-mice, whereas antibody levels increased in nontolerant animals that rejected porcine thymus grafts. These data show that porcine thymic xenotransplantation can induce donor-specific tolerance in immunocompetent hu-mice, supporting this approach for tolerance induction in clinical xenotransplantation.

in vivo T cell depletion, in vivo prevention of graft rejection

Tonomura N, Shimizu A, Wang S, Yamada K, Tchipashvili V, Weir GC, Yang YG. (2008). "Pig islet xenograft rejection in a mouse model with an established human immune system" Xenotransplantation 15(2):129-35. PubMed

Background: Xenotransplantation from pigs provides a potential solution to the severe shortage of human pancreata, but strong immunological rejection prevents its clinical application. A better understanding of the human immune response to pig islets would help develop effective strategies for preventing graft rejection. Methods: We assessed pig islet rejection by human immune cells in humanized mice with a functional human immune system. Humanized mice were prepared by transplantation of human fetal thymus/liver tissues and CD34(+) fetal liver cells into immunodeficient mice. Islet xenograft survival/rejection was determined by histological analysis of the grafts and measurement of porcine C-peptide in the sera of the recipients. Results: In untreated humanized mice, adult pig islets were completely rejected by 4 weeks. These mice showed no detectable porcine C-peptide in the sera, and severe intra-graft infiltration by human T cells, macrophages, and B cells, as well as deposition of human antibodies. Pig islet rejection was prevented by human T-cell depletion prior to islet xenotransplantation. Islet xenografts harvested from T-cell-depleted humanized mice were functional, and showed no human cell infiltration or antibody deposition. Conclusions: Pig islet rejection in humanized mice is largely T-cell-dependent, which is consistent with previous observations in non-human primates. These humanized mice provide a useful model for the study of human xenoimmune responses in vivo.

in vitro inhibition of MLR

Xu Y, Kolber-Simonds D, Hope JA, Bazin H, Latinne D, Monroy R, White-Scharf ME, Schuurman HJ. (2004). "The anti-CD2 monoclonal antibody BTI-322 generates unresponsiveness by activation-associated T cell depletion" Clin Exp Immunol 138(3):476-83. PubMed

The antihuman CD2 MoAb BTI-322 (Lo-CD2a) effectively inhibits T cell responses in vitro to allogeneic cells, which is followed by unresponsiveness to the original stimulator in secondary stimulation. We studied the xenogeneic human antiporcine mixed lymphocyte reaction (MLR), and utilized anti-T cell receptor (TCR) Vbeta family antibody-induced cell proliferation to determine the specificity and mechanism. BTI-322 and its humanized version, MEDI-507, effectively inhibited the primary xenogeneic MLR. After suboptimal primary stimulation using lower numbers of xenogeneic stimulator cells, the unresponsiveness in secondary culture was apparent only for xenogeneic stimulator cells of the original SLA haplotype, and not for third-party stimulators or allogeneic cells. The inhibition of primary MLR was not observed for nylon-wool-purified T cells, but was seen after reconstitution of purified T cells with monocytes. Similarly, anti-Vbeta family-specific stimulation showed family-specific unresponsiveness in secondary culture. This required the presence of the whole BTI-322 molecule: a F(ab')2 fragment was not effective. T cells of a distinct Vbeta family were depleted after stimulation with an anti-Vbeta family-specific antibody and BTI-322. We conclude that the inhibition by BTI-322 of a primary xenogeneic MLR or the response to an anti-TCR Vbeta antibody is associated with unresponsiveness upon restimulation, due to activation-associated cell depletion. In this process, the interaction between monocytes and the Fc part of the antibody is involved. This unique characteristic of BTI-322 suggests the potential of the antibody for tolerance induction in vivo, besides the potential use as a T cell depleting agent.

in vivo prevention of graft rejection, Functional Assays

Snanoudj R, Rouleau M, Bidère N, Carmona S, Baron C, Latinne D, Bazin H, Charpentier B, Senik A. (2004). "A role for CD2 antibodies (BTI-322 and its humanized form) in the in vivo elimination of human T lymphocytes infiltrating an allogeneic human skin graft in SCID mice: an Fcgamma receptor-related mechanism involving co-injected human NK cells" Transplantation 78(1):50-8. PubMed

Background: Pilot clinical studies have shown that the rat anti-human-CD2 monoclonal antibody, LoCD2a/BTI-322, can efficiently prevent and treat acute kidney rejection. However, the in vivo mechanism by which it prevents allograft rejection has not been studied. BTI-322 and its humanized form have been shown to mediate in vitro antibody-dependent cell-mediated cytotoxicity (ADCC) against CD2 cells through the activation of monocytes or natural killer (NK) cells. Methods: Human fetal skin samples were grafted into severe combined immunodeficient/nonobese diabetic mice. Five weeks later (day 0), the mice were injected with human allogeneic peripheral blood lymphocytes (PBL). Either on day 0 or on day 14, mice were treated with BTI-322, hu-BTI-322, or their F(ab')2 fragments. Peripheral blood mononuclear cells (PBMC) thoroughly devoid of NK cells were also assayed. Results: After injection of PBL, the human skins became heavily infiltrated with activated human T lymphocytes, resulting in dermal microvascular injuries indicative of graft rejection. Early treatment with BTI-322 and hu-BTI-322 prevented all these events. These CD2 antibodies rapidly eliminated human T lymphocytes that had already infiltrated the grafts, with no evidence of recirculation toward the spleen. Their F(ab')2 fragments were, in contrast, ineffective. Elimination of NK cells from injected PBMC prevented the curative effect exerted by whole CD2 antibodies. It also abrogated their cytotoxicity potential against CD2 cells in ADCC assays. Conclusion: F(ab')2 fragments of the CD2 antibodies could not prevent allograft rejection, whereas whole immunoglobulin G could, and human NK cells were required for the curative effect exerted by these antibodies. The results are consistent with an FcgammaR-dependent ADCC mechanism mediated in vivo by human NK cells.

in vivo prevention of graft rejection, Functional Assays, ELISA

Nizet Y, Chentoufi AA, de la Parra B, Lewalle P, Rouas R, Cornet A, Besse T, Mourad M, Malaise J, Squifflet JP, Bazin H, Latinne D. (2000). "The experimental (in vitro) and clinical (in vivo) immunosuppressive effects of a rat IgG2b anti-human CD2 mAb, LO-CD2a/BTI-322" Transplantation 69(7):1420-8. PubMed

Background: CD2 is a cell surface glycoprotein expressed on most human T cells and natural killer (NK) cells, working as a cell adhesion and costimulatory molecule. The aim of this paper is to analyze the mechanism of action of a rat IgG2b anti-human CD2 monoclonal antibody (mAb) (LO-CD2a/BTI-322 mAb), which is a potent immunosuppressive agent and inducer of cell death. In vivo, this mAb is able to prevent or treat kidney allograft rejection. Methods: The mechanisms by which the LO-CD2a/BTI-322 mAb is able to induce inhibition of cell activation and cell death were analyzed by mixed lymphocyte reactions and by flow cytometry. After in vivo treatment, levels of circulating mAb were measured by ELISA as well as anti-rat immunization and cytokine release. Results: We show that the inhibition of cell activation induced by LO-CD2a/BTI-322 mAb after allogeneic or OKT3 stimulation is due to an Fcgamma receptor-dependent CD2 down-modulation and to T-cell depletion through an antibody-dependent cell-mediated cytotoxicity mechanism mediated by NK cells or activated monocytes. Peripheral T- and NK-cell depletion was observed after in vivo treatment with LO-CD2a/BTI322. Cytokine release (TNFalpha) was correlated with some side effects, but only after the first injection, and the effects were never severe or life threatening. Conclusion: The correlation between the in vitro and in vivo data suggests that T-cell depletion, especially of activated cells, and inhibition of cell activation after CD2 down-modulation are the main mechanisms of action of the LO-CD2a/BTI-322 mAb.

in vitro inhibition of MLR

Xu Y, Ryan D, Wu C, Bazin H, Latinne D, White-Scharf ME, Thall AD. (2000). "Inhibition of human anti-pig T cell response by anti-CD2, anti-CD40L, and CTLA4-Ig: a comparative study" Transplant Proc 32(5):926. PubMed

in vitro inhibition of MLR

Branco L, Barren P, Mao SY, Pfarr D, Kaplan R, Postema C, Langermann S, Koenig S, Johnson S. (1999). "Selective deletion of antigen-specific, activated T cells by a humanized MAB to CD2 (MEDI-507) is mediated by NK cells" Transplantation 68(10):1588-96. PubMed

CD2 is a 50-kDa transmembrane glycoprotein that plays an important role in T and natural killer (NT) lymphocyte functions. CD2 serves as both an adhesion molecule and as a costimulatory molecule through interactions with its ligand, CD58, on antigen presenting or target cells. Consistent with earlier studies using a rat anti-CD2 mAb, we have shown that treatment of alloantigen stimulated T lymphocytes with a humanized mAb, MEDI-507 (IgG1, kappa), induced hyporesponsiveness to subsequent stimulation with alloantigen but not to mitogen (phytohemagglutinin). Fluorescence-activated cell sorting analysis of cells from mixed lymphocyte reaction (MLR) treated with MEDI-507 revealed pronounced deletion of T and NK cells, consistent with lack of proliferation in the MLR. MEDI-507 F(ab')2 fragments did not have inhibitory activity or induce deletion of lymphocytes in the MLR. Removal of the NK cell subset by magnetic bead depletion using anti-CD16 and anti-CD56 mAbs eliminated both the T cell deletion and the inhibitory effect. Reconstitution of NK depleted responder populations using autologous NK cells restored the MEDI-507-mediated deletion activity to levels measured in the original MLR. Formaldehyde-fixed NK cells failed to mediate the MEDI-507-induced deletion effect. Altogether, our studies indicate that activated T cells with MEDI-507 bound to CD2 are preferential targets for autologous NK cells through a nonapoptotic cytotoxic mechanism.

Functional Assays

Nizet Y, Chentoufi AA, Havaux X, Kinet I, Cormont F, Bazin H, Latinne D. (1999). "Apoptosis of human naive NK cells mediated by a rat IgG2b anti CD2 mAb through a fractricidal ADCC reaction" Immunol Lett 68(2-3):229-35. PubMed

LO-CD2a/BTI-322, a rat anti human CD2 mAb, shows in vitro and in vivo immunosuppressive properties and induces T-cell depletion resulting partially from an antibody dependent cellular cytotoxicity (ADCC) mediated by NK cells. The aim of this paper is to study the in vitro effect of LO-CD2a/BTI-322 on NK cells, the majority of them also expressing the CD2 molecule. The addition of the mAb to purified naive NK cells induces apoptosis of CD2+ cells. The apoptosis is rapid, Fas ligand independent and completely inhibited by the calcium chelator EGTA, suggesting a fractricidal ADCC reaction and implying that NK cells are not resistant to lysis when used as target cells. At the end of the reaction, the CD2 - remaining cells are still capable of natural cytotoxicity against K562 cells, but at a lower rate than untreated cells.

Functional Assays

Dumont C, Déas O, Mollereau B, Hebib C, Giovino-Barry V, Bernard A, Hirsch F, Charpentier B, Senik A. (1998). "Potent apoptotic signaling and subsequent unresponsiveness induced by a single CD2 mAb (BTI-322) in activated human peripheral T cells" J Immunol 160(8):3797-804. PubMed

Manipulation of CD2 molecules with CD2 mAb pairs has been shown to deliver apoptotic signals to activated mature T cells. We show that BTI-322, a CD2 mAb directed at a peculiar epitope of CD2, can trigger on its own the apoptotic death of IL-2-activated peripheral T cells and of OKT3-stimulated T cells, contrasting in this respect with a series of other mouse or rat CD2 mAb. F(ab')2 fragments were as potent as the whole Ab. BTI-322-induced apoptosis proceeded in a few hours and was independent of the Fas/Fas ligand system. Less than 5 ng/ml of BTI-322, added at the beginning of culture, were able to eliminate within 4 days most CD3+ cells from OKT3- and IL-2-stimulated lymphocytes, the only cells remaining being CD16+CD2- NK cells. T cell proliferative responses induced by a mitogenic CD2 mAb pair or by PHA-P (which mainly binds to CD2) were not inhibited by BTI-322. In this case, the apoptotic effect was successfully counteracted by simultaneous enhancement of T cell divisions. Thus, the killing effect of BTI-322 was most effective when T cells were exclusively stimulated through the CD3/TCR complex. Apoptosis of the responding T cells may explain why T cells recovered from a primary MLC performed in the presence of BTI-322 responded to third party cells but not to the primary stimulatory cells. These data constitute the rational basis for the use of BTI-322 for inducing tolerance in human allotransplantation.

in vivo prevention of graft rejection, Functional Assays, ELISA

Przepiorka D, Phillips GL, Ratanatharathorn V, Cottler-Fox M, Sehn LH, Antin JH, LeBherz D, Awwad M, Hope J, McClain JB. (1998). "A phase II study of BTI-322, a monoclonal anti-CD2 antibody, for treatment of steroid-resistant acute graft-versus-host disease" Blood 92(11):4066-71. PubMed

BTI-322, a rat monoclonal IgG2b directed against the CD2 antigen on T cells and natural killer (NK) cells, blocks primary and memory alloantigen proliferative responses in vitro. We have evaluated the pharmacokinetics and safety of BTI-322 during treatment of 20 transplant recipients with steroid-refractory acute graft-versus-host disease (GVHD). Treatment consisted of BTI-322 by intravenous (IV) bolus or 30-minute infusion at approximately 0.1 mg/kg/d for 10 days in addition to continuing high-dose steroids and tacrolimus or cyclosporine. Pharmacokinetic sampling was performed in 10 patients; the t1/2 +/- SE was 9.1 +/- 1.3 hours, the Cmax was 2,549 +/- 291 ng/mL, the Vd was 3.97 +/- 0.95 L, and the Vd/kg was 0. 05 +/- 0.01 L/kg. Ten patients experienced transient dyspnea sometimes accompanied by nausea, vomiting, diarrhea, and tachycardia shortly after the initial bolus dose of drug, but serious drug-related adverse events were not seen during the remainder of the infusions. At the end of treatment (day 11), there were six patients with complete responses and five with a reduction in grade of GVHD for a total response rate of 55% (95% confidence interval [CI], 32% to 77%). Antibodies targeting CD2 may be active in the treatment of acute GVHD, and evaluation of a humanized form of BTI-322 is warranted.

in vivo prevention of graft rejection

Squifflet JP, Besse T, Malaise J, Mourad M, Delcorde C, Hope JA, Pirson Y. (1997). "BTI-322 for induction therapy after renal transplantation: a randomized study" Transplant Proc 29(1-2):317-9. PubMed

in vivo prevention of graft rejection

Mourad M, Besse T, Malaise J, Baldi A, Latinne D, Bazin H, Pirson Y, Hope J, Squifflet JP. (1997). "BTI-322 for acute rejection after renal transplantation" Transplant Proc 29(5):2353. PubMed

in vivo prevention of graft rejection

Besse T, Malaise J, Mourad M, Pirson Y, Hope J, Awwad M, White-Scharf M, Squifflet JP. (1997). "Prevention of rejection with BTI-322 after renal transplantation (results at 9 months)" Transplant Proc 29(5):2425-6. PubMed

in vivo prevention of graft rejection, Functional Assays

Latinne D, De La Parra B, Nizet Y, Cornet A, Giovino-Barry V, Monroy RL, White-Scharf ME, Bazin H. (1996). "An anti-CD2 mAb induces immunosuppression and hyporesponsiveness of CD2+ human T cells in vitro" Int Immunol 8(7):1113-9. PubMed

We describe here the potent specific immunosuppression obtained in vitro by LO-CD2a, a rat mAb directed against the human CD2 molecule. Addition of low dose LO-CD2a (40 ng/ml) at the time of mixed lymphocyte culture (MLC) initiation inhibits 80% of the proliferation and, more impressive, addition of the mAb 4 days after culture initiation at a similar concentration still suppresses 50% of the MLC. When responder T cells previously treated with LO-CD2a are challenged a second time by the same donor or third party allogeneic cells, hyporesponsiveness occurs in both cases, although reactivity to T cell mitogenic stimulation persists. Finally, the low production of cytokines such as tumor necrosis factor-alpha and IFN-gamma after incubation of human T cells with LO-CD2a suggests the absence of T cell activation. These results demonstrate that LO-CD2a mAb has a significant immunosuppressive effect and induces hyporesponsiveness in vitro, thereby suggesting potential efficacy in vivo for the treatment of acute rejection and for the induction of tolerance in allotransplantation.

in vivo prevention of graft rejection, Immunohistochemistry (frozen)

Bombil F, Kints JP, Havaux X, Scheiff JM, Bazin H, Latinne D. (1995). "A rat monoclonal anti-(human CD2) and L-leucine methyl ester impacts on human/SCID mouse graft and B lymphoproliferative syndrome" Cancer Immunol Immunother 40(6):383-9. PubMed

The transfer of human peripheral blood mononuclear cells (hu-PBMC) from adult Epstein-Barr-virus(EBV)-seropositive donors in SCID (severe combined immunodeficiency) mice frequently leads to the development of a human B lymphoproliferative syndrome (hu-BLPS). Therefore, as 90% of adult potential donors are EBV-seropositive, efforts have to be made to avoid the occurrence of this B lymphoproliferative disorder. McCune et al. [Science 241:1632 (1988)] used human fetal organs for a human SCID graft. This system does not give rise to hu-BLPS but human fetal organs are much less available than peripheral blood leucocytes. The experiments reported in this paper show how crucial is the presence of functional T lymphocytes for a graft to take and for development of hu-BLPS in hu-PBMC-reconstituted SCID mice, since inhibition of T lymphocyte by a rat anti-(human CD2) monoclonal antibody (LO-CD2a) during the first 10 days of the graft prevents successful engraftment of human normal lymphocytes as well as hu-BLPS in SCID mice. The transfer of B cells alone or B cells plus monocytes in SCID mice does not permit either long-term engraftment or development of hu-BLPS. We also demonstrate that hu-PBMC treated with L-leucine methyl ester are less susceptible to the development of hu-BLPS after engraftment in SCID mice than are untreated hu-PBMC. The mechanism of action of L-leucine methyl ester on these cells is discussed.