Preparation of a Growth Hormone Receptor/Prolactin Receptor Bispecific Antibody Antagonist Which Exhibited Anti-Cancer Activity

Prolactin receptor (PRLR) and growth hormone receptor (GHR) are closely related to the occurrence and development of breast cancer, and breast cancer cell endogenously express GHR, PRLR and GHR-PRLR heterodimer. In this case, the combined use of PRLR or GHR inhibitors may produce better anti-breast cancer potential than PRLR or GHR inhibitors alone. In this case, it is necessary to develop the dual-function GHR/PRLR antagonists with anti-breast cancer potential. For this, we used hybridoma technology to generate an anti-idiotypic antibody (termed H53). We then used various techniques, including competitive ELISA, competitive receptor binding analysis, and indirect immunofluorescence assay to identify H53, and the results show that H53 behaves as a typical internal image anti-idiotypic antibody (Ab2β). Further experiments indicate that H53 is a dual-function inhibitor, which not only inhibited PRLR-mediated intracellular signaling, but also blocked GHR-mediated intracellular signaling in a dose-dependent manner. Furthermore, H53 could inhibit PRL/GH-driven cancer cell proliferation in vivo and in vitro. This study indicates that H53 exhibits potential biological activity against breast tumors, which implies that internal image anti-idiotypic antibodies may be a useful strategy for the development of PRLR/GHR dual-function antagonists for breast cancer therapy.

MYH9 Key Amino Acid Residues Identified by the Anti-Idiotypic Antibody to Porcine Reproductive and Respiratory Syndrome Virus Glycoprotein 5 Involve in the Virus Internalization by Porcine Alveolar Macrophages

MYH9 has been identified as an indispensable cellular protein for porcine reproductive and respiratory syndrome virus (PRRSV) entry into permissive cells using the monoclonal anti-idiotypic antibody (Mab2-5G2) recognizing an antibody that specifically interacts with PRRSV glycoprotein 5 (GP5). More recently, we found that Mab2-5G2 interacted with the MYH9 C-terminal domain, designated PRA, which is required for PRRSV internalization. In this study, we demonstrate that blocking of MYH9 with Mab2-5G2 significantly diminished PRRSV internalization by porcine alveolar macrophage (PAM) via interruption of direct interaction between GP5 and MYH9, and thus remarkably inhibited subsequent infection of PAMs by PRRSV-2 isolates. Moreover, the three-dimensional structure of the Mab2-5G2 Fab-PRA complex determined via homology modeling predicted potential docking sites required for PRRSV internalization. Further analysis of Mab2-5G2-binding sites within PRA highlighted that the amino acids E1670, K1673, E1679, and I1683 in PRA are the key Mab2-5G2-binding residues. Notably, recombinant PRA protein blocked the interaction between PRRSV GP5 and cellular MYH9 by preventing translocation of MYH9 from the cytoplasm to the cell membrane, an essential step for PRRSV virion internalization. Meanwhile, porcine cell line permissive for PRRSV bearing point mutation of E1670A in MYH9 demonstrated reduced susceptibility for PRRSV infection. In conclusion, this work increases understanding of both PRRSV pathogenesis and the mechanistic role played by MYH9 in PRRSV infection.

Antibody humanization—the Influence of the antibody framework on the CDR-H3 loop ensemble in solution

Antibody engineering of non-human antibodies has focused on reducing immunogenicity by humanization, being a major limitation in developing monoclonal antibodies. We analyzed four series of antibody binding fragments (Fabs) and a variable fragment (Fv) with structural information in different stages of humanization to investigate the influence of the framework, point mutations and specificity on the complementarity determining region (CDR)-H3 loop dynamics. We also studied a Fv without structural information of the anti-idiotypic antibody Ab2/3H6, because it completely lost its binding affinity upon superhumanization, as an example of a failed humanization. Enhanced sampling techniques in combination with molecular dynamics simulations allow to access micro- to milli-second timescales of the CDR-H3 loop dynamics and reveal kinetic and thermodynamic changes involved in the process of humanization. In most cases, we observe a reduced conformational diversity of the CDR-H3 loop when grafted on a human framework and find a conformational shift of the dominant CDR-H3 loop conformation in solution. A shallow side minimum of the conformational CDR-H3 loop ensemble attached to the murine framework becomes the dominant conformation in solution influenced by the human framework. Additionally, we observe in the case of the failed humanization that the potentially binding competent murine CDR-H3 loop ensemble in solution shows nearly no kinetical or structural overlap with the superhumanized variant, thus explaining the loss of binding.

Anti-idiotypic antibodies elicit anti-HIV-1–specific B cell responses

Human anti-HIV-1 broadly neutralizing antibodies (bNAbs) protect against infection in animal models. However, bNAbs have not been elicited by vaccination in diverse wild-type animals or humans, in part because B cells expressing the precursors of these antibodies do not recognize most HIV-1 envelopes (Envs). Immunogens have been designed that activate these B cell precursors in vivo, but they also activate competing off-target responses. Here we report on a complementary approach to expand specific B cells using an anti-idiotypic antibody, iv8, that selects for naive human B cells expressing immunoglobulin light chains with 5–amino acid complementarity determining region 3s, a key feature of anti-CD4 binding site (CD4bs)–specific VRC01-class antibodies. In mice, iv8 induced target cells to expand and mature in the context of a polyclonal immune system and produced serologic responses targeting the CD4bs on Env. In summary, the results demonstrate that an anti-idiotypic antibody can specifically recognize and expand rare B cells that express VRC01-class antibodies against HIV-1.

Is Tumorigenesis an Abiogenesis?

CLN-IgG (Pritumuab) was subjected to clinical trials aiming at regression of brain tumors. The mechanism underlying the dramatic recuperation of cancer patient was considered by means of augmentation of idiotypic antibody-mediated internal image transmission of the vimentin epitope-vipidam. Silencing of prionogenicity of vimentin by chaperonic antibody CLN-IgG was a pertinent modality to elongate cancer patient's senescence with little side effect.