4-1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, ...and IFNγ production. The ability to induce potent antitumor activity by stimulating 4-1BB on tumor-specific cytotoxic T cells makes 4-1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4-1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4-1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4-1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4-1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4-1BB-mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors.
Prostate cancer is the most common cancer and the second leading cause of cancer deaths among males in most Western countries. Autologous cellular immunotherapy for the treatment of cancer seeks to ...induce tumor-specific immunity in the patient and is consequently dependent on a suitable target antigen and effective presentation of that antigen to the patient's immune system. Prostatic acid phosphatase (PAP) has been tested as a target antigen due to its high and apparently specific expression in the prostate. We used a variety of approaches to analyze PAP expression, including immunohistochemistry, in situ hybridization, and quantitative polymerase chain reaction. We complemented these laboratory-based techniques with an in silico analysis of reported PAP expression in human cDNA libraries. Our studies confirmed that, while PAP expression is not restricted to prostate tissues, its expression in other human tissues is approximately 1-2 orders of magnitude less than that observed in the prostate. The relative specificity of PAP expression in the prostate supports its use as a target of autologous cellular immunotherapy. The approach described here, involving the use of multiple correlates of tissue-specific expression, is warranted as a prerequisite in selecting any suitable target for immunotherapy.
Under most circumstances, allelic exclusion at the T cell receptor (TCR)β locus is tightly regulated. Here, we describe a system in which TCRβ allelic exclusion is overcome as a result of V(D)J ...recombination in peripheral CD4
+ T cells. In TCR β chain transgenic mice, tolerogen-mediated chronic peripheral selection against cells expressing the transgene leads to surface expression of endogenous TCR β chains. Peripheral CD4
+ T cells reexpress the recombination activating genes,
RAG1 and
RAG2, and contain signal end intermediates indicative of ongoing V(D)J recombination. The rescue from deletion of mature T cells expressing newly generated TCR β chains suggests that receptor revision plays a role in the maintenance of peripheral T cell tolerance.
Interleukin-1 alpha and -1 beta (IL-1$\alpha $ and IL-1$\beta $) are cytokines that participate in the regulation of immune responses, inflammatory reactions, and hematopoiesis. A direct expression ...strategy was used to clone the receptor for IL-1 from mouse T cells. The product of the cloned complementary DNA binds both IL-1$\alpha $ and IL-1$\beta $ in a manner indistinguishable from that of the native T cell IL-1 receptor. The extracellular, IL-1 binding portion of the receptor is 319 amino acids in length and is composed of three immunoglobulin-like domains. The cytoplasmic portion of the receptor is 217 amino acids long.
Recent studies have demonstrated that DNA cleavage during V(D)J recombination is mediated by the RAG1 and RAG2 proteins. These proteins must therefore bind to the recombination signals, but the ...specific binding interaction has been difficult to study in vitro. Here, we use an in vivo one-hybrid DNA binding assay to demonstrate that RAG1, in the absence of RAG2, can mediate signal recognition via the nonamer, with the heptamer acting to enhance its binding. A region of RAG1 with sequence similarity to bacterial invertases is essential for DNA binding. Localization of RAG2 to the signal is dependent upon the presence of RAG1 and is substantially more efficient with a 12 bp spacer signal than with a 23 bp spacer signal.
► Through TCR revision, autoreactive TCRs are replaced by those generated through post-thymic DNA rearrangement. ► Downmodulation of surface TCR expression initiates RAG re-expression and TCR ...revision. ► In Vβ5 transgenic mice, loss of surface TCR is not mediated by transgene loss or diminished expression. ► Downregulation of surface TCR expression occurs in two stages, only one of which requires tolerogen expression.
TCR revision is a tolerance mechanism by which self-reactive TCRs expressed by mature CD4
+ peripheral T cells are replaced by receptors encoded by genes generated by post-thymic DNA rearrangement. The downmodulation of surface TCR expression initiates TCR revision, and serves as a likely trigger for the induction of the recombinase machinery. We show here in a Vβ5 transgenic mouse model system that downregulation of the self-reactive transgene-encoded TCR is not maintained by transgene loss or diminished transcription or translation. The downregulation of surface TCR expression likely occurs in two stages, only one of which requires tolerogen expression.
Abstract
We have characterized a subset of CD8 T cells (CD8 Treg) with immunosuppressive characteristics in inflammatory disease settings. Here we describe the binding and specificity profiles of a ...novel bispecific CD8 Treg modulator in vitro, ex vivo, and in vivo, and initial assessment of tolerability and pharmacology in a humanized mouse model.
We evaluated the binding, pharmacokinetics (PK), and early tolerability of the bispecific CD8 Treg modulator targeting CD8 and KIR2DL1/2/3. Despite single arm binding to NK cells via the KIR targeting arm and CD8 T cells by the CD8 targeting arm of the bispecific, we did not detect activation of immune cells or increased pro-inflammatory cytokines in vitro, nor did we observe any contribution of NK cells to the elimination of pathogenic CD4 T cells. In healthy mice, the CD8 Treg modulator had a PK profile consistent with antibody molecules; treatment delayed disease onset in an acute GVHD setting. In humanized CD34-engrafted NSG-IL-15 transgenic mice, supporting engraftment of NK cells, no activation of immune cells in peripheral blood or terminal tissues or induction of proinflammatory cytokines in the serum was observed following a single dose of the CD8 Treg modulator.
The recently described CD8 Treg network appears dysfunctional in autoimmune diseases. The KIRxCD8 targeting bispecific modulator targets this network, engages and activates CD8 Treg and reduces inflammation without increasing unwanted immune cell activation or pro-inflammatory cytokines, both in vitro and in vivo. Our early data suggest that KIR-mediated enhancement of CD8 Treg function using a bispecific modulator is a broadly applicable and a promising CD8 Treg specific therapeutic modality for the treatment of autoimmune disease.
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