Target Expression, Generation, Preclinical Activity, and Pharmacokinetics of the BCMA-T Cell Bispecific Antibody EM801 for Multiple Myeloma Treatment.

We identified B cell maturation antigen (BCMA) as a potential therapeutic target in 778 newly diagnosed and relapsed myeloma patients. We constructed an IgG-based BCMA-T cell bispecific antibody (EM801) and showed that it increased CD3 T cell/myeloma cell crosslinking, followed by CD4/CD8 T cell activation, and secretion of interferon-γ, granzyme B, and perforin. This effect is CD4 and CD8 T cell mediated.

Scaffold hopping towards potent and selective JAK3 inhibitors: discovery of novel C-5 substituted pyrrolopyrazines.

The discovery of a novel series of pyrrolopyrazines as JAK inhibitors with comparable enzyme and cellular activity to tofacitinib is described. The series was identified using a scaffold hopping approach aided by structure based drug design using principles of intramolecular hydrogen bonding for conformational restriction and targeting specific pockets for modulating kinase activity.

Discovery of a series of novel 5H-pyrrolo[2,3-b]pyrazine-2-phenyl ethers, as potent JAK3 kinase inhibitors.

We report the discovery of a novel series of ATP-competitive Janus kinase 3 (JAK3) inhibitors based on the 5H-pyrrolo[2,3-b]pyrazine scaffold. The initial leads in this series, compounds 1a and 1h, showed promising potencies, but a lack of selectivity against other isoforms in the JAK family. Computational and crystallographic analysis suggested that the phenyl ether moiety possessed a favorable vector to achieve selectivity.

Strategic use of conformational bias and structure based design to identify potent JAK3 inhibitors with improved selectivity against the JAK family and the kinome.

Using a structure based design approach we have identified a series of indazole substituted pyrrolopyrazines, which are potent inhibitors of JAK3. Intramolecular electronic repulsion was used as a strategy to induce a strong conformational bias within the ligand. Compounds bearing this conformation participated in a favorable hydrophobic interaction with a cysteine residue in the JAK3 binding pocket, which imparted high selectivity versus the kinome and improved selectivity within the JAK family.

3-Amido pyrrolopyrazine JAK kinase inhibitors: development of a JAK3 vs JAK1 selective inhibitor and evaluation in cellular and in vivo models.

The Janus kinases (JAKs) are involved in multiple signaling networks relevant to inflammatory diseases, and inhibition of one or more members of this class may modulate disease activity or progression. We optimized a new inhibitor scaffold, 3-amido-5-cyclopropylpyrrolopyrazines, to a potent example with reasonable kinome selectivity, including selectivity for JAK3 versus JAK1, and good biopharmaceutical properties. Evaluation of this analogue in cellular and in vivo models confirmed functional selectivity for modulation of a JAK3/JAK1-dependent IL-2 stimulated pathway over a JAK1/JAK2/Tyk2-dependent IL-6 stimulated pathway.

RN486, a selective Bruton's tyrosine kinase inhibitor, abrogates immune hypersensitivity responses and arthritis in rodents.

Genetic mutation and pharmacological inhibition of Bruton's tyrosine kinase (Btk) both have been shown to prevent the development of collagen-induced arthritis (CIA) in mice, providing a rationale for the development of Btk inhibitors for treating rheumatoid arthritis (RA). In the present study, we characterized a novel Btk inhibitor, 6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one (RN486), in vitro and in rodent models of immune hypersensitivity and arthritis. We demonstrated that RN486 not only potently and selectively inhibited the Btk enzyme, but also displayed functional activities in human cell-based assays in multiple cell types, blocking Fcε receptor cross-linking-induced degranulation in mast cells (IC(50) = 2.

Immunostimulatory Tim-1-specific antibody deprograms Tregs and prevents transplant tolerance in mice.

T cell Ig mucin (Tim) molecules modulate CD4(+) T cell responses. In keeping with the view that Tim-1 generates a stimulatory signal for CD4(+) T cell activation, we hypothesized that an agonist Tim-1-specific mAb would intensify the CD4(+) T cell-dependant allograft response. Unexpectedly, we determined that a particular Tim-1-specific mAb exerted reciprocal effects upon the commitment of alloactivated T cells to regulatory and effector phenotypes.

OX40 controls functionally different T cell subsets and their resistance to depletion therapy.

T cell depletion is a widely used approach in clinical transplantation. However, not all T cells are equally sensitive to depletion therapies and a significant fraction of T cells persists even after aggressive treatment. The functional attributes of such T cells and the mechanisms responsible for their resistance to depletion are poorly studied.

OX40 costimulation turns off Foxp3+ Tregs.

OX40 is a recently identified T-cell costimulatory molecule that belongs to the TNF/TNFR superfamily. OX40 can be expressed by both activated T effector cells and Foxp3(+) Tregs. It is well known that OX40 delivers a potent costimulatory signal to T effector cells, but very little is known about the role of OX40 in regulating the suppressor properties of Foxp3(+) Tregs and the de novo generation of new inducible Foxp3(+) Tregs from T effector cells.

NK cells promote transplant tolerance by killing donor antigen-presenting cells.

Natural killer (NK) cells are programmed to kill target cells without prior antigen priming. Because of their potent cytolytic activities, NK cells are one of the key cell types involved in dismantling allografts. However, in certain transplant models, NK cells also express potent immunoregulatory properties that promote tolerance induction.

Critical, but conditional, role of OX40 in memory T cell-mediated rejection.

Memory T cells can be a significant barrier to the induction of transplant tolerance. However, the molecular pathways that can regulate memory T cell-mediated rejection are poorly defined. In the present study we tested the hypothesis that the novel alternative costimulatory molecules (i.

Study of T-cell costimulatory blockade in vivo at a single-cell level.

The critical role of costimulatory signals in T-cell activation and the complexity of T-cell costimulatory pathways involved make a detailed understanding of this system a challenging task. By taking advantage of the unique chemical properties of CFSE, we and others have developed an in vivo model that allows quantitative analysis of T-cell activation at a single-cell level. This model involves labeling of donor T-cells with the tracking dye CFSE and adoptively transferring into lethally irradiated allogeneic hosts.

The common gammac-cytokines and transplantation tolerance.

Transplant rejection, like tolerance, is a T cell-dependent event. There is compelling evidence to suggest that induction of transplant tolerance is an actively learned process in which T cells need to engage the alloantigens in order to learn to tolerate the allograft. A family of cytokines whose receptors use the same IL-2 receptor gamma chain (also called the common gammac) plays an important role in regulating multiple aspects of the allograft response (i.

Different costimulatory and growth factor requirements for CD4+ and CD8+ T cell-mediated rejection.

Costimulatory signals and growth factor signals play a key role in commanding T cell activation and T cell effector function. However, how costimulatory signals and growth factor signals interact and integrate into the activation program of CD4(+) and CD8(+) T cells during the allograft response remains poorly defined. In the present study we found that either CD4- or CD8-deficient mice can vigorously reject the skin allografts.

Combination therapy of malononitrilamide FK778 with tacrolimus on cell proliferation assays and in rats receiving renal allografts.

Background: Malononitrilamide FK778, an analogue of leflunomide's active metabolite, is a promising novel small molecule with immunosuppressive and immunomodulatory properties. In this study, we evaluated the ability of combination therapy of FK778 with tacrolimus to inhibit lymphocyte proliferation and to prevent acute allograft rejection.

Methods: Proliferation assay was used to evaluate the effect of FK778 plus tacrolimus on murine splenocytes, monkey lymphocytes, and human peripheral blood mononuclear cells, after activation with T or B cell-specific mitogens.

Synergistic effect of vincristine with tacrolimus or sirolimus in prevention of acute heart allograft rejection in the rat.

The application of multiple immunosuppressive therapy for organ transplantation could enhance therapeutic efficacy, while minimizing the toxicity of individual drugs used in the regimen. In this study, the effect of the combined therapy of vincristine (VCR) with tacrolimus (FK506) or sirolimus (rapamycin, RAPA) was tested in prevention of acute heart allograft rejection in the rat. A Brown Norway (BN, RT 1(n)) to Lewis (LEW, RT 1(1)) rat combination was used in a heart allografting model.

Significant prolongation of renal allograft survival by delayed combination therapy of FK778 with tacrolimus in nonhuman primates.

Background: Malononitrilamide 715 (FK778) is a new class of low-molecular-weight immunosuppressant that is a derivative of the active metabolite of leflunomide, A77 1726. In this study, the authors evaluated the combined effect of FK778 with tacrolimus in prevention of renal allograft rejection in Vervet monkeys.

Methods: Male Vervet monkeys were obtained from Caribbean Primates Ltd.

Renal ischemia-reperfusion injury in the rat is prevented by a novel immune modulation therapy.

Background: Vasogen Inc.'s (Mississauga, Ontario, Canada) immune modulation therapy (IMT) is a therapy in which cells from the patient's own blood are modified by ex vivo exposure to specific physicochemical stressors, including oxidation, ultraviolet (UV) light, and an elevated temperature. The therapy has been shown to have a beneficial effect in models of inflammation and vascular diseases.

Restoration of fertility in oophorectomized rats after tubo-ovarian transplantation.

Improved microsurgical techniques for en bloc vascularized adnexal isograft in the rat is described. The graft of the right ovary together with its fallopian tube and upper third of uterus was transplanted orthotopically with end-to-side anastomoses between the donor aortic segment and recipient aorta and between the donor vena cava segment and recipient inferior vena cava, with end-to-end anastomosis of the donor and recipient uterus in a syngeneic, bilaterally oophorectomized rat. All transplantations were successful in terms of immediate vascular patency rate (10/10, 100%).