The ligand for CD40 (CD40L) is a membrane glycoprotein on activated T cells that induces B cell proliferation and immunoglobulin secretion. Abnormalities in the CD40L gene were associated with an X-linked immunodeficiency in humans [hyper-IgM (immunoglobulin M) syndrome]. This disease is characterized by elevated concentrations of serum IgM and decreased amounts of all other isotypes. CD40L complementary DNAs from three of four patients with this syndrome contained distinct point mutations. Recombinant expression of two of the mutant CD40L complementary DNAs resulted in proteins incapable of binding to CD40 and unable to induce proliferation or IgE secretion from normal B cells. Activated T cells from the four affected patients failed to express wild-type CD40L, although their B cells responded normally to wild-type CD40L. Thus, these CD40L defects lead to a T cell abnormality that results in the failure of patient B cells to undergo immunoglobulin class switching.
Abstract CD69 is a disulfide‐linked homo‐dimer expressed on the surface of activated T cells, B cells, natural killer cells, neutrophils and platelets. Antibody cross‐linking of CD69 in the presence of phorbol ester results in cellular activation events including proliferation and the induction of specific genes. Using an expression cloning strategy we have isolated cDNA encoding human CD69 from a CD4 + T cell clone. Transfection of the cDNA clone in CV‐1/EBNA cells results in the expression of a covalently linked homodimer. The cDNA insert hybridizes to a 1.7‐kb mRNA in phorbol 12‐myristate 13‐acetate‐ or phytohemoagglutinin‐stimulated human T cells. Using the human clone we have isolated cDNA encoding mouse CD69, which, when expressed in human T cells allowed those cells to respond to anti‐mouse CD69 antibodies by secreting interleukin‐2 and interferon‐γ. Sequence analysis showed that both mouse and human CD69 are type II membrane glycoproteins related to the NKR‐P1 and Ly‐49 families of natural killer cell activation molecules.
Antibody–drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two separate maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the observed efficacy was similar. More severe thrombocytopenia was observed with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approximately 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance observed for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equivalent doses and exacerbation of clinical pathology parameters (decreased tolerability) at equivalent doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index.
We have expressed the CD40 surface Ag as both a soluble 28-kDa molecule and a 57-kDa Fc fusion protein containing the human IgG1 Fc region. Soluble CD40 and the Fc fusion protein inhibited the proliferative response of anti-IgM-activated human B cells to the CD40 mAb G28-5. Similarly, G28-5- and IL-4-induced IgE secretion from PBMC depleted of T cells was effectively blocked by both forms of soluble CD40. Although the soluble constructs of CD40 had only a minimal inhibitory effect on IL-4-mediated proliferation of anti-IgM-activated B cells, IL-4-induced soluble CD23 shedding from both PBMC and T cells depleted of PBMC, and IgE secretion from PBMC, were significantly reduced in a concentration-dependent manner when soluble CD40 was present in the culture. The data presented demonstrate that both soluble forms of the CD40 molecule are biologically active, and suggest that the ligand for CD40 is inducible in IL-4-stimulated cultures and that it mediates both shedding of sCD23 and IgE secretion.
