SummaryThe first immunoglobulin V-like domain of CD4 contains the binding site for human immunodeficiency virus gp120. Guided by the atomic structure of a two-domain CD4 fragment, we have examined gp120 interaction with informative CD4 mutants, both by equilibrium and kinetic analysis. The binding site on CD4 appears to be a surface region of about 900 A 2 on the C" edge of the domain. It contains an exposed hydrophobic residue, Phe43, on the C" strand and four positively charged residues, Lys29, Lys35, LYS46, and Arg59, on the C, C', C", and D strands, respectivdy. Replacement of Phe43 with Ala or Ile reduces affinity for gp120 by more than 500-fold; Tyr, Trp, and Leu substitutions have smaller effects. The four positively charged side chains each make significant contributions (7-50-fold). This CD4 site may dock into a conserved hydrophobic pocket bordered by several negatively charged residues in gpi20. Class II major histocompatibility complex binding includes the same region on CD4; this overlap needs to be considered in the design of inhibitors of the CD4-gp120 interaction.T he CD4 transmembrane glycoprotein participates in key steps of thymocyte differentiation (1-3) and T lymphocyte activation (4-9) by interacting with nonpolymorphic regions of class II MHC molecules (10--12). It is expressed on most thymocytes (1) and a subset of T lymphocytes, including helper T cells and class II MHC-specific cytotoxic T cells (5-7, D, 14). CD4 in humans is also the receptor for HIV (15-18). It is required for viral attachment and subsequent entry into ceils (19), and these functions account for HIV T cell tropism. The virus binds through a contact made by the gp120 component of its envelope glycoprotein (20). The binding of gp120 to CD4 has been implicated in the cellular depletion of CD4 + lymphocytes in AIDS (21-24), and circulating free gp120 is thought to interfere with normal immune function (12, 25). The central role of the CD4-gp120 interaction in the pathogenesis of AIDS justifies a detailed analysis in molecular terms, and inhibition of that interaction is also an attractive target for antiviral intervention.The extracellular part of CD4 contains four Ig-like do= mains (26). The gp120 binding site lies in the most NH2 terminal of these domains (27,28). A number of studies eraploying escape mutants (29), insertion mutagenesis (30), homology scanning mutagenesis (12,28,31), and alanine scanning mutagenesis (32) have helped to restrict the binding site to a segment surrounding the region homologous to the CDR2 of an Ig V domain.The atomic structure of a fragment containing the first two domains of CD4 has been determined by x-ray crystallography (33, 34). The first domain contains two antiparallel sheets, as predicted by its Ig V homology. The mutations that affect gp120 binding lie mostly in the antiparalld strands, denoted C' and C", that form a ridge at one edge of the domain as well as in the adjacent D strand. The C'C" loop is longer than the CDR2 loop of a typical Ig V domain, and it projects prominently ...
Purpose: 4-1BB (CD137) is a key costimulatory immunoreceptor and promising therapeutic target in cancer. To overcome limitations of current 4-1BB-targeting antibodies, we have developed PRS-343, a 4-1BB/HER2 bispecific molecule. PRS-343 is designed to facilitate T-cell costimulation by tumorlocalized, HER2-dependent 4-1BB clustering and activation. Experimental Design: PRS-343 was generated by the genetic fusion of 4-1BB-specific Anticalin proteins to a variant of trastuzumab with an engineered IgG4 isotype. Its activity was characterized using a panel of in vitro assays and humanized mouse models. The safety was assessed using ex vivo human cell assays and a toxicity study in cynomolgus monkeys. Results: PRS-343 targets 4-1BB and HER2 with high affinity and binds both targets simultaneously. 4-1BB-expressing T cells are efficiently costimulated when incubated with PRS-343 in the presence of cancer cells expressing HER2, as evidenced by increased production of proinflammatory cytokines (IL2, GM-CSF, TNFa, and IFNg). In a humanized mouse model engrafted with HER2-positive SK-OV-3 tumor cells and human peripheral blood mononuclear cells, PRS-343 leads to tumor growth inhibition and a dose-dependent increase of tumor-infiltrating lymphocytes. In IND-enabling studies, PRS-343 was found to be well tolerated, with no overt toxicity and no relevant drug-related toxicologic findings. Conclusions: PRS-343 facilitates tumor-localized targeting of T cells by bispecific engagement of HER2 and 4-1BB. This approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity compared with current monospecific approaches. The reported data led to initiation of a phase I clinical trial with this first-in-class molecule. See related commentary by Su et al., p. 5732
Human CD8+ lymphocyte subpopulations were analyzed for their expression of CD8 alpha and CD8 beta subunits. Investigations with uncloned peripheral blood lymphocytes as well as cloned human natural killer and T cell subpopulations demonstrate that CD3- natural killer cells, T cell receptor gamma/delta, and CD4+CD8+ T cell clones express exclusively CD8 alpha gene products. Structural analysis of CD8 molecules demonstrates that CD8 alpha+/beta- T lymphocytes surface express 75-kDa CD8 alpha/alpha homodimers whereas CD8 alpha/beta lymphocytes express concomittantly two CD8 isoforms of different molecular masses (67 kDa and 75 kDa, respectively). Peptide mapping of these latter two isoforms suggests that CD8 is expressed as alpha/alpha homodimers and alpha/beta heterodimers on CD8 alpha/beta+ cells. Importantly, we found that the two CD8 isoforms behave functionally different. Thus, in contrast to CD8 alpha/beta+/CD8 alpha/alpha+ T lymphocytes, cytolytic activity of CD8 alpha/beta-/CD8 alpha/alpha+ T cell clones was not inhibited by anti-CD8 monoclonal antibodies and the latter were not induced to proliferate following CD3/CD8 cross-linking.
Recent work has identified L1CAM (CD171) as a novel marker for human carcinoma progression. Functionally, L1CAM promotes tumor cell invasion and motility, augments tumor growth in nude mice, and facilitates experimental tumor metastasis. These functional features qualify L1 as an interesting target molecule for tumor therapy. Here, we generated a series of novel monoclonal antibodies (mAb) to the L1CAM ectodomain that were characterized by biochemical and functional means. All novel mAbs reacted specifically with L1CAM and not with the closely related molecule CHL1, whereas antibodies to the COOH terminal part of L1CAM (mAb2C2, mAb745H7, pcytL1) showed cross-reactivity. Among the novel mAbs, L1-9.3 was selected and its therapeutic potential was analyzed in various isotype variants in a model of SKOV3ip cells growing i.p. in CD1 nude mice. Only therapy with the IgG2a variant efficiently prolonged survival and reduced tumor burden. This was accompanied by an increased infiltration of F4/80-positive monocytic cells. Clodronate pretreatment of tumor-bearing animals led to the depletion of monocytes and abolished the therapeutic effect of L1-9.3/IgG2a. Expression profiling of tumor-derived mRNA revealed that L1-9.3/IgG2a therapy induced altered expression of cellular genes associated with apoptosis and tumor growth. Our results establish that anti-L1 mAb therapy acts via immunologic and nonimmunologic effector mechanism to block tumor growth. The novel antibodies to L1CAM could become helpful tools for the therapy of L1-positive human carcinomas.
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