From Rosette receptor to CD2
In 1971, while investigating the interaction between antigen–antibody–complement complexes and lymphoid cells, Lay et al (1) noticed that sheep red blood cells (RBC) adhered to a surprisingly large proportion of human peripheral blood lymphocytes forming clusters or “rosettes”.
This immuno-assay, now referred as erythrocyte rosetting or E-rosetting, demonstrates the number of D-positive cells in a D-negative suspension using an anti-D reagent. The anti-D binds to D-positive fetal RBCs, and when indicator D-positive RBCs are added, rosettes are formed. The rosette test is now a screening test for Fetal-Maternal Hemorrhage (FMH) that detects fetal D+ red cells in maternal Rh negative blood (Nancy L. Van Buren MD, in Transfusion Medicine and Hemostasis, 2019).
In 1981, Kamoun et al (2) reported that a specific monoclonal antibody, designated 9.6, linked an unknown 50kDa surface protein that appears to be present on all E-rosette-forming cells. The discovered structure was named the E rosette receptor.
The E rosette receptor, is a transmembrane protein for thymocytes, peripheral T cells, NK cells, and a subset of thymic B cells, acting in accessory signal generation and transduction (3). Having been branded and renamed multiple times under the designations T-cell surface antigen T11/Leu-5, LFA-2, T11, SRBC, the E-rosette receptor is now known under the name Cluster of Differentiation 2 or CD2.
Receptor structure and natural ligands of the glycoprotein CD2
This glycoprotein of 327 amino acids (351 amino acids including signal sequence), part of the immunoglobulin superfamily, is the receptor of lymphocyte-associated antigen 3 (LFA3; also known as CD58, and major ligand of CD2), CD48 (low affinity), CD15s (Sialyl Lewis X) and CD59.
While CD2 is broadly expressed on murine B cells, human CD2 expression is restricted to a limited number of B cell subtypes, like B-cell neoplasia. Normal CD2+ B-cell populations are observed in adults and may represent the nonmalignant counterpart of CD2+ B-cell neoplasms (4). Further complicating the tasks of researchers in in-vivo studies, mice don’t express LFA3, a cell surface protein expressed mainly on antigen-presenting cells.
The extracellular domain of CD2 (amino acids 25–209) of two immunoglobulin-like domains and three glycosylation sites (N89, N141, and N150) is linked to a highly conserved proline-rich cytoplasmic tail via a transmembrane helix.
Thanks to its structure, CD2 is the essential element that connects T and NK cells on one side, and antigen-presenting cells on the other, being so an active element of the immunological synapse.
This cell-cell junction discovered by Abraham Kupferis composed of several transmembrane proteins (CD2, LFA-1, CD4, CD8, ICAM-1, CD28, et CD80/CD86) that form a kind of ring around the T receptor, in which each interacts with its respective partner.
Immunological effects of the CD2 immunoglobulin superfamily on T and NK cells
Membrane-bound ligands like CD58 or CD48 activates CD2-mediated signaling in the absence of TCR triggering (through the actin-dependent coalescence of signaling molecules TCR-ζ chain, Lck, and LAT), and enhances T cell signaling additively with TCR (5).
CD2 expression is upregulated on memory T cells compared to naïve T cells in humans (6) and cell activation caused a 1.5-fold increase in the number of CD2 sites on the cell surface, and increase the affinity of CD2 for CD58 by 2.5-fold (7) relative to resting T cells. This unique feature represents an opportunity for the develoment of treatments for transplant and autoimmune disorders.
Moreover, CD2 signals also contribute to the continuous expansion of CD28−CD8+ T cells during chronic stimulation by persistent antigen (8) offering possibilities in infectious diseases.
Similar to T cells, the response of natural killer (NK) cells is regulated through the reorganization of actin cytoskeleton and the formation of immunological synapse (NKIS) where CD2 plays an important role.
In fact, CD2 has been identified as a key co-stimulatory receptor (9), which contributes to increased cytokine production in adaptive NK cells after synergising with CD16 (also known as Fc γ receptor III; FcγRIII).
Therapeutic Applications of SYnAbs anti-CD2 monoclonal antibodies
From SYnAbs LO-CD2a to Siplizumab
SYnAbs first developed a rat-LOU anti-CD2 monoclonal antibody. This monoclonal recognizes all mature T cells, 95% of thymocytes, most NK cells, but no B cells. This anti-CD2a antibody (LO-CD2a, BTI-322) prevents and treats allograft rejection in experimental models (10). It has been used in human renal transplantation, in the treatment of graft-versus-host disease after marrow transplantation, in liver transplantation (11), in plaque psoriasis (12), transplant induction therapy (13, 14), stem cell transplantation (15), graft-vs.-host disease (16) and T cell malignancies (17).
SYnAbs licensed this therapeutic asset to MedImmune (now AstraZeneca). The rebranded MEDI-507 has been humanized and this IgG1 antibody format is now known as Siplizumab.
LO-CD2a, however, does not react with baboon or cynomolgus monkey T cells and therefore cannot be evaluated in tolerance experimental settings. SYnAbs has therefore developed a new mAb (LO-CD2b) that shows similar properties to LO-CD2a but is active on both primate and human CD2+ cells.
SYnAbs LO-CD2b : a nonactivating rat anti-CD2 mAb able to strongly inhibit both mitogenic and allogeneic responses in humans and non human primates
LO-CD2b has been characterized by flow cytometry, E-rosetting inhibition, and Western blotting.
In vitro inhibition of immune responses by LO-CD2b was assessed after both mitogenic and allogeneic stimulation in mixed lymphocyte reactions (MLR). Several LO-CD2b dose and time responses were tested. In vivo, peripheral and lymph node T-cell depletion was examined both by flow cytometry and immunohistology in 10 baboons that received intravenous injection of LO-CD2b at different doses and time courses. Xenosensitization (anti-rat) was assessed by ELISA. Renal allograft survival was followed in two baboons treated with iterative LO-CD2b injections.
Administration of 200 ng/ml LO-CD2b almost completely inhibited human and baboon mitogenic stimulation. Allogeneic baboon and human MLR were completely inhibited by the addition of LO-CD2b (at 312 ng/ml) on the day of the initiation of culture; when added after 1 or 2 days, LO-CD2b still provided a significant MLR inhibition (>50%). Incubation of LO-CD2b with baboon peripheral blood mononuclear cells produced very low cytokine levels (interferon-γ, tumor necrosis factor-α, interleukin 2).
In secondary MLR, baboon peripheral blood mononuclear cells previously incubated with LO-CD2b were unable to respond to a second allogeneic stimulation but were able to react to mitogens. In vivo, within the first hour after LO-CD2b injection (at 0.15, 0.5, and 2 mg/kg), an 85–90% peripheral depletion of CD2+ cells was observed. A partial T-cell depletion in inguinal lymph nodes was seen after 1 week. The mechanism of peripheral T-cell depletion could have been antibody-dependent cell cytotoxicity or opsonization but was complement independent. Iterative LO-CD2b injections (12 days at 0.35 mg/kg) slightly prolonged the renal allograft survival in two baboons.
LO-CD2b is a nonactivating rat anti-CD2 mAb able to strongly inhibit both mitogenic and allogeneic responses in human and nonhuman primate.
In vivo, LO-CD2b provides a rapid peripheral T-cell depletion, which is reversible within days after the cessation of injections.
This rat mAb represents a very important asset for therapeutic applications (18).
Thanks to its unique platform, SYnAbs has been able to generate a new anti-IL-2R monoclonal antibody (LO-Tact-1), produced in the LOU/C rat species, available for clinical use with T depletion strategy. This asset, with the same MoA as SiplizumAb (LO-CD2a, MEDI-507) previously developed by SYnAbs, shows cross-reactivity to human and cynomolgus CD2. LO-CD2b IgG2bκ rat-LOU monoclonal antibody has been tested into several in-vitro, ex-vivo and in-vivo studies as described below.
Specificity to CD2 transmembrane receptor
1. Competitive assay: Serial dilutions of LO-CD2b were incubated with baboon PBMCs and then with rhodamine-labeled T11, Leu5b (anti-CD2) mAbs. The percentage of rhodamine-labeled cells was assessed by FC, thereby demonstrating a specific competition for the CD2 molecule.
2. E-rosetting: LO-CD2b blocked E-rosetting at an average of 91±2% for LO-CD2b-coated cells (data not shown)
3. Western blot: 125I anti-rat sheep Ig binding to (A) IgG2b rat mAb as a positive control; (B) IgG1b mouse Ig and (C) no reagent as negative controls; (D) supernatant of lysed PBMC+LO-CD2b. LO-CD2b is reactive with a lysed PBMC supernatant of a molecular weight of 52 kDa.
LO-CD2 inhibition of mitogenic stimulation
When LO-CD2b was added at day 0, the phytohemagglutinin (PHA) stimulation of baboon PBMCs was inhibited at 72.8±12.1%, and the concanavalin (ConA) stimulation at 71.0±8.4%.
LO-CD2b was still able to partially inhibit a mitogenic stimulation after the culture initiation: the addition of LO-CD2b at 1 or 2 days after culture initiation inhibited the PHA stimulation at 56±8.5% and 38.5±9.8%, respectively, and inhibited the ConA stimulation at 29.3±7.5% and 15.7±11.5%, respectively.
LO-CD2b had a similar inhibitor effect on PHA-stimulated human PBMCs. In the presence of human PBMCs, the inhibition after PHA stimulation was 62.1% at day 0, 48% at day 1, and 18% at day 2.
Inhibition of allogeneic one-way MLR
When added at the day of initiation of culture, a LO-CD2b concentration more than 156 ng/ml almost completely (96.4±1.1%) inhibited a baboon allogeneic MLR. Low doses such as 4.8 and 9.7 ng/ml inhibited 50% of the proliferative response, and 1.2 ng/ml of LO-CD2b inhibited the MLR up to 22.9±14.1%.
The same effect was observed in allogeneic human MLR but with a (10–20%) lower degree of inhibition. In comparison, no inhibition was obtained with a rat IgG2b isotype control (data not shown).
REFERENCES
(1) LAY, W., MENDES, N., BIANCO, C. et al. Binding of Sheep Red Blood Cells to a Large Population of Human Lymphocytes. Nature 230, 531–532 (1971). https://doi.org/10.1038/230531a0
(2) Kamoun M, Martin PJ, Hansen JA, Brown MA, Siadak AW, Norwinski RC. Identification of a human T lymphocyte surface protein associated with the E-rosette receptor. J Exp Med 1981;153:207–304.
(3) Suthanthiran M. T-cell differentiation antigen cluster 2 (CD2) is a receptor for accessory cells and can generate and/or transduce accessory signals. Cell Immunol. 1988 Mar;112(1):112-22. doi: 10.1016/0008-8749(88)90280-8. PMID: 3257905.
(4) Kingma DW, Imus P, Xie XY, Jasper G, Sorbara L, Stewart C, Stetler-Stevenson M. CD2 is expressed by a subpopulation of normal B cells and is frequently present in mature B-cell neoplasms. Cytometry. 2002 Oct 15;50(5):243-8. doi: 10.1002/cyto.10131. PMID: 12360573.
(5) Yoshihisa Kaizuka, Adam D. Douglass, Santosh Vardhana, Michael L. Dustin, Ronald D. Vale; The coreceptor CD2 uses plasma membrane microdomains to transduce signals in T cells. J Cell Biol 4 May 2009; 185 (3): 521–534. doi: https://doi.org/10.1083/jcb.200809136
(6) Sanders ME, Makgoba MW, Sharrow SO, Stephany D, Springer TA, Young HA, Shaw S. Human memory T lymphocytes express increased levels of three cell adhesion molecules (LFA-3, CD2, and LFA-1) and three other molecules (UCHL1, CDw29, and Pgp-1) and have enhanced IFN-gamma production. J Immunol. 1988 Mar 1;140(5):1401 -7. PMID: 2894392.
(7) Zhu D-M, Dustin ML, Cairo CW, Thatte HS, Golan DE. Mechanisms of cellular avidity regulation in CD2-CD58-Mediated T Cell adhesion. ACS Chem Biol. (2006) 1:649–58. doi: 10.1021/cb6002515
(8) Leitner J, Herndler-Brandstetter D, Zlabinger GJ, Grubeck-Loebenstein B. CD58/CD2 is the primary costimulatory pathway in human CD28-CD8 T cells. J Immunol. (2015) 195:477–87. doi: 10.4049/jimmunol.1401917
(9) Liu LL, Landskron J, Ask EH, Enqvist M. Critical role of CD2 Co-stimulation in adaptive natural killer cell responses revealed in NKG2C-deficient humans. Cell Rep. (2016) 15:1088–99. doi: 10.1016/j.celrep.2016.04.005
(10) Snanoudj R., Rouleau M., Bidere N., Carmona S., Baron C. et al. — 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, 2004, 78, 50-58.
(11) Lerut J., Van Thuyne V., Mathijs J., Lemaire J., Talpe S. et al. — Anti-CD2 monoclonal antibody and tacrolimus in adult liver transplantation. Transplantation, 2005, 80, 1186-1193.
(12) Langley RG, Papp K, Bissonnette R, Toth D. Safety profile of intravenous and subcutaneous siplizumab, an anti-CD2 monoclonal antibody, for the treatment of plaque psoriasis: results of two randomized, double-blind, placebo-controlled studies. Int J Dermatol. (2010) 49:818–28. doi: 10.1111/j.1365-4632.2010.04512.x
(13) Koenecke C, Shaffer J, Alexander SI, Preffer F. NK cell recovery, chimerism, function, and recognition in recipients of haploidentical hematopoietic cell transplantation following nonmyeloablative conditioning using a humanized anti-CD2 mAb, Medi-507. Exp Hematol. (2003) 31:911–23. doi: 10.1016/S0301-472X(03)00224-8
(14) Pruett TL, McGory RW, Wright FH, Pescovitz MD. Safety profile, pharmacokinetics, and pharmacodynamics of siplizumab, a humanized anti-CD2 monoclonal antibody, in renal allograft recipients. Transplant Proc. (2009) 41:3655–61. doi: 10.1016/j.transproceed.2009.06.226
(15) Spitzer TR, McAfee SL, Dey BR, Colby C. Nonmyeloablative haploidentical stem-cell transplantation using anti-CD2 monoclonal antibody (MEDI-507)-based conditioning for refractory hematologic malignancies. Transplantation. (2003) 75:1748–51. doi: 10.1097/01.TP.0000064211.23536.AD
(16) Adkins D, Ratanatharathorn V, Yang H, White B. Safety profile and clinical outcomes in a phase I, placebo-controlled study of siplizumab in acute graft-versus-host disease. Transplantation. (2009) 88:198–202. doi: 10.1097/TP.0b013e3181abfbf7
(17) Roswarski J, Roschewski M, Lucas A, Melani C. Phase I dose escalation study of the anti-CD2 monoclonal antibody, siplizumab, with DA-EPOCH-R in aggressive peripheral T-cell lymphomas. Leukemia Lymphoma. (2018) 59:1466–9. doi: 10.1080/10428194.2017.1387908
(18) Dehoux, Jean-Paul; Talpe, Stéphanie; Dewolf, Natacha; Otsuka, Masayuki; Oike, Fumitaka; Jamar, François; de la Parra, Bernardo; Latinne, Dominique; Bazin, Hervé; Gianello, Pierre. « EFFECTS ON HUMAN AND NONHUMAN PRIMATE IMMUNE RESPONSE OF A NEW RAT ANTI-CD2 MONOCLONAL ANTIBODY » iBooks.
