Selective depletion of tumor-infiltrating regulatory T cells with BAY 3375968, a novel Fc-optimized anti-CCR8 antibody.

Regulatory T cells (Tregs) are known to facilitate tumor progression by suppressing CD8+ T cells within the tumor microenvironment (TME), thereby also hampering the effectiveness of immune checkpoint inhibitors (ICIs). While systemic depletion of Tregs can enhance antitumor immunity, it also triggers undesirable autoimmune responses. Therefore, there is a need for therapeutic agents that selectively target Tregs within the TME without affecting systemic Tregs. In this study, as shown also by others, the chemokine (C-C motif) receptor 8 (CCR8) was found to be predominantly expressed on Tregs within the TME of both humans and mice, representing a unique target for selective depletion of tumor-residing Tregs. Based on this, we developed BAY 3375968, a novel anti-human CCR8 antibody, along with respective surrogate anti-mouse CCR8 antibodies, and demonstrated their in vitro mode-of-action through induction of potent antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities. In vivo, anti-mouse CCR8 antibodies effectively depleted Tregs within the TME primarily via ADCP, leading to increased CD8+ T cell infiltration and subsequent tumor growth inhibition across various cancer models. This monotherapeutic efficacy was significantly enhanced in combination with ICIs. Collectively, these findings suggest that CCR8 targeting represents a promising strategy for Treg depletion in cancer therapies. BAY 3375968 is currently under investigation in a Phase I clinical trial (NCT05537740).

T cell-mediated elimination of cancer cells by blocking CEACAM6-CEACAM1 interaction.

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a cell surface receptor, is expressed on normal epithelial tissue and highly expressed in cancers of high unmet medical need, such as non-small cell lung, pancreatic, and colorectal cancer. CEACAM receptors undergo homo- and heterophilic interactions thereby regulating normal tissue homeostasis and angiogenesis, and in cancer, tumor invasion and metastasis. CEACAM6 expression on malignant plasma cells inhibits antitumor activity of T cells, and we hypothesize a similar function on epithelial cancer cells. The interactions between CEACAM6 and its suggested partner CEACAM1 on T cells were studied. A humanized CEACAM6-blocking antibody, BAY 1834942, was developed and characterized for its immunomodulating effects in co-culture experiments with T cells and solid cancer cells and in comparison to antibodies targeting the immune checkpoints programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and T cell immunoglobulin mucin-3 (TIM-3). The immunosuppressive activity of CEACAM6 was mediated by binding to CEACAM1 expressed by activated tumor-specific T cells. BAY 1834942 increased cytokine secretion by T cells and T cell-mediated killing of cancer cells. The in vitro efficacy of BAY 1834942 correlated with the degree of CEACAM6 expression on cancer cells, suggesting potential in guiding patient selection. BAY 1834942 was equally or more efficacious compared to blockade of PD-L1, and at least an additive efficacy was observed in combination with anti-PD-1 or anti-TIM-3 antibodies, suggesting an efficacy independent of the PD-1/PD-L1 axis. In summary, CEACAM6 blockade by BAY 1834942 reactivates the antitumor response of T cells. This warrants clinical evaluation.

Rational Design of Single Copy Expression Cassettes in Defined Chromosomal Sites Overcomes Intraclonal Cell-to-Cell Expression Heterogeneity and Ensures Robust Antibody Production.

The expression of endogenous genes as well as transgenes depends on regulatory elements within and surrounding genes as well as their epigenetic modifications. Members of a cloned cell population often show pronounced cell-to-cell heterogeneity with respect to the expression of a certain gene. To investigate the heterogeneity of recombinant protein expression we targeted cassettes into two preselected chromosomal hot-spots in Chinese hamster ovary (CHO) cells. Depending on the gene of interest and the design of the expression cassette, we found strong expression variability that could be reduced by epigenetic modifiers, but not by site-specific recruitment of the modulator dCas9-VPR. In particular, the implementation of ubiquitous chromatin opening elements (UCOEs) reduced cell-to-cell heterogeneity and concomitantly increased expression. The application of this method to recombinant antibody expression confirmed that rational design of cell lines for production of transgenes with predictable and high titers is a promising approach.

Modular Protein Expression Toolbox (MoPET), a standardized assembly system for defined expression constructs and expression optimization libraries.

The design and generation of an optimal expression construct is the first and essential step in in the characterization of a protein of interest. Besides evaluation and optimization of process parameters (e.g. selection of the best expression host or cell line and optimal induction conditions and time points), the design of the expression construct itself has a major impact. However, the path to this final expression construct is often not straight forward and includes multiple learning cycles accompanied by design variations and retesting of construct variants, since multiple, functional DNA sequences of the expression vector backbone, either coding or non-coding, can have a major impact on expression yields. To streamline the generation of defined expression constructs of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed. This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules with a minimal cloning burden. Combining these features with a standardized cloning strategy facilitates the identification of optimized DNA expression constructs in shorter time. The MoPET system currently consists of 53 defined DNA modules divided into eight functional classes and can be flexibly expanded. However, already with the initial set of modules, 792,000 different constructs can be rationally designed and assembled. Furthermore, this starting set was used to generate small and mid-sized combinatorial expression optimization libraries. Applying this screening approach, variants with up to 60-fold expression improvement have been identified by MoPET variant library screening.

The prion-like domain in the exomer-dependent cargo Pin2 serves as a trans-Golgi retention motif.

Prion and prion-like domains (PLDs) are found in many proteins throughout the animal kingdom. We found that the PLD in the S. cerevisiae exomer-dependent cargo protein Pin2 is involved in the regulation of protein transport and localization. The domain serves as a Pin2 retention signal in the trans-Golgi network (TGN). Pin2 is localized in a polarized fashion at the plasma membrane of the bud early in the cell cycle and the bud neck at cytokinesis. This polarized localization is dependent on both exo- and endocytosis. Upon environmental stress, Pin2 is rapidly endocytosed, and the PLD aggregates and causes sequestration of Pin2. The aggregation of Pin2 is reversible upon stress removal and Pin2 is rapidly re-exported to the plasma membrane. Altogether, these data uncover a role for PLDs as protein localization elements.

Evaluation of human pancreatic RNase as effector molecule in a therapeutic antibody platform.

Human antibody-ribonuclease (RNase) fusion proteins, referred to as immunoRNases, have been proposed as an alternative to heterologous immunotoxins, without their immunogenicity and unspecific toxicity issues. In this study, we investigated if human pancreatic RNase will be suitable as effector component in a therapeutic antibody development platform. We generated several fusion proteins consisting of tumor-specific human immunoglobulins (IgGs) and human pancreatic RNase. Transient mammalian cell production was efficient and IgG-RNases were purified to homogeneity. Antigen binding was comparable to the parental antibodies and RNase catalytic activity was retained even in the presence of 50-fold molar excess of human cytosolic RNase inhibitor (RI). Serum stability, cell binding and internalization of IgG-RNases were comparable to the parental IgGs. Despite these promising properties, none of the IgG-RNases revealed significant inhibition of tumor cell growth in vitro even when targeting different antigens putatively employing different endocytotic pathways. The introduction of different linkers containing endosomal protease cleavage sites into the IgG-RNase did not enhance cytotoxicity. Similarly, RI evasive human pancreatic RNase variants mediated only small inhibiting effects on tumor cell growth at high concentrations, potentially reflecting inefficient cytosolic translocation. Taken together, human pancreatic RNase and variants did not prove to be generally suitable as effector component for a therapeutic antibody drug development platform.

Transport to the plasma membrane is regulated differently early and late in the cell cycle in Saccharomyces cerevisiae.

Traffic from the trans-Golgi network to the plasma membrane is thought to occur through at least two different independent pathways. The chitin synthase Chs3p requires the exomer complex and Arf1p to reach the bud neck of yeast cells in a cell-cycle-dependent manner, whereas the hexose transporter Hxt2p localizes over the entire plasma membrane independently of the exomer complex. Here, we conducted a visual screen for communalities and differences between the exomer-dependent and exomer-independent transport to the plasma membrane in Saccharomyces cerevisiae. We found that most of the components that are required for the fusion of transport vesicles with the plasma membrane, are involved in localization of both Chs3p and Hxt2p. However, the lethal giant larva homologue Sro7p is required primarily for the targeting of Chs3p, and not Hxt2p or other cargoes such as Itr1p, Cwp2p and Pma1p. Interestingly, this transport defect was more pronounced in large-budded cells just before cytokinesis than in small-budded cells. In addition, we found that the yeast Rab11 homologue Ypt31p determines the residence time of Chs3p in the bud neck of small-budded, but not large-budded, cells. We propose that transport to and from the bud neck is regulated differently in small- and large-budded cells, and differs early and late in the cell cycle.

TOR1 and TOR2 have distinct locations in live cells.

TOR is a structurally and functionally conserved Ser/Thr kinase found in two multiprotein complexes that regulate many cellular processes to control cell growth. Although extensively studied, the localization of TOR is still ambiguous, possibly because endogenous TOR in live cells has not been examined. Here, we examined the localization of green fluorescent protein (GFP) tagged, endogenous TOR1 and TOR2 in live S. cerevisiae cells. A DNA cassette encoding three copies of green fluorescent protein (3XGFP) was inserted in the TOR1 gene (at codon D330) or the TOR2 gene (at codon N321). The TORs were tagged internally because TOR1 or TOR2 tagged at the N or C terminus was not functional. The TOR1(D330-3XGFP) strain was not hypersensitive to rapamycin, was not cold sensitive, and was not resistant to manganese toxicity caused by the loss of Pmr1, all indications that TOR1-3XGFP was expressed and functional. TOR2-3XGFP was functional, as TOR2 is an essential gene and TOR2(N321-3XGFP) haploid cells were viable. Thus, TOR1 and TOR2 retain function after the insertion of 748 amino acids in a variable region of their noncatalytic domain. The localization patterns of TOR1-3XGFP and TOR2-3XGFP were documented by imaging of live cells. TOR1-3XGFP was diffusely cytoplasmic and concentrated near the vacuolar membrane. The TOR2-3XGFP signal was cytoplasmic but predominately in dots at the plasma membrane. Thus, TOR1 and TOR2 have distinct localization patterns, consistent with the regulation of cellular processes as part of two different complexes.

Arf1p, Chs5p and the ChAPs are required for export of specialized cargo from the Golgi.

In Saccharomyces cerevisiae, the synthesis of chitin is temporally and spatially regulated through the transport of Chs3p (chitin synthase III) to the plasma membrane in the bud neck region. Traffic of Chs3p from the trans-Golgi network (TGN)/early endosome to the plasma membrane requires the function of Chs5p and Chs6p. Chs6p belongs to a family of four proteins that we have named ChAPs for Chs5p-Arf1p-binding Proteins. Here, we show that all ChAPs physically interact not only with Chs5p but also with the small GTPase Arf1p. A short sequence at the C-terminus of the ChAPs is required for protein function and the ability to bind to Chs5p. Simultaneous disruption of two members, Deltabud7 and Deltabch1, phenocopies a Deltachs6 or Deltachs5 deletion with respect to Chs3p transport. Moreover, the ChAPs interact with each other and can form complexes. In addition, they are all at least partially localized to the TGN in a Chs5p-dependent manner. Most importantly, several ChAPs can interact physically with Chs3p. We propose that the ChAPs facilitate export of cargo out of the Golgi.

New modules for the repeated internal and N-terminal epitope tagging of genes in Saccharomyces cerevisiae.

Epitope tagging is a powerful method for the rapid analysis of protein function. In Saccharomyces cerevisiae epitope tags are introduced easily into chromosomal loci by homologous recombination using a simple PCR-based strategy. Although quite a number of tools exist for C-terminal tagging as well as N-terminal tagging of proteins expressed by heterologous promoters, there are only very limited possibilities to tag proteins at the N-terminus and retain the endogenous expression level. Furthermore, no PCR-templates for internal tagging have been reported. Here we describe new modules that are suitable for both the repeated N-terminal and internal tagging of proteins, leaving their endogenous promoters intact. The tags include 6xHA, 9xMyc, yEGFP, TEV-GST-6xHIS, ProtA, TEV-ProtA and TEV-ProtA-7xHIS in conjunction with different heterologous selection markers.

Arf1p provides an unexpected link between COPI vesicles and mRNA in Saccharomyces cerevisiae.

The small GTPase Arf1p is involved in different cellular processes that require its accumulation at specific cellular locations. The recruitment of Arf1p to distinct points of action might be achieved by association of Arf1p with different proteins. To identify new interactors of Arf1p, we performed an affinity chromatography with GTP- or GDP-bound Arf1p proteins. A new interactor of Arf1p-GTP was identified as Pab1p, which binds to the polyA-tail of mRNAs. Pab1p was found to associate with purified COPI-coated vesicles generated from Golgi membranes in vitro. The stability of the Pab1p-Arf1p complex depends on the presence of mRNA. Both symmetrically distributed mRNAs as well as the asymmetrically localized ASH1 mRNA are found in association with Arf1p. Remarkably, Arf1p and Pab1p are both required to restrict ASH1 mRNA to the bud tip. Arf1p and coatomer play an unexpected role in localizing mRNA independent and downstream of the SHE machinery. Hereby acts the SHE machinery in long-range mRNA transport, whereas COPI vesicles could act as short-range and localization vehicles. The endoplasmic reticulum (ER)-Golgi shuttle might be involved in concentrating mRNA at the ER.

Policy perspectives on public health for Mexican migrants in California.

This analysis focuses on public policies that affect primary HIV prevention and access to HIV care for Mexican migrants residing in California. Policy or structural level interventions, as opposed to behavioral or psychologic interventions, help to shape the environment in which people live. We use a conceptual model for policy analysis in public health to understand better the challenges faced by Mexican migrants. We assess potential policy level interventions that may serve as barriers to or facilitators of primary HIV prevention and care for Mexican migrants. Among potential barriers, we discuss restrictions on public health services based on legal immigration status, limits placed on affirmative action in education, and laws limiting travel and immigration. Under potential facilitators, we discuss community and migrant health centers, language access laws, and the use of community-based groups to provide prevention and treatment outreach. We also report on the limited research evaluating the implications of these public policies and ways to organize for more responsive public policies.