Search

Aim: We report clinical activity and safety of sitravatinib in patients with advanced cancer from basket cohorts with specific molecular alterations, in a Phase Ib study. Materials & methods: Patients with advanced solid tumors harboring amplification, mutation, or rearrangement of MET , AXL , RET , NTRK , DDR2 , KDR , PDGFRA , KIT or CBL received sitravatinib once daily. Primary end point was confirmed objective response rate (ORR). Results: In total, 113 patients were enrolled following a median of 3 (range 1-18) prior systemic regimens. Altered RET (n = 31), CBL (n = 31) and MET (n = 17) were most frequent cohorts. Overall, 68.9% had reduced tumor volume and most (61.5%) had a best objective response of stable disease. ORR was highest in patients with RET -rearranged non-small cell lung cancer (21.1%) but did not differ significantly from the null hypothesis (ORR ≤15%; p = 0.316). Median progression-free survival and overall survival (5.7 and 24.2 months, respectively) were also longest in the RET -rearranged non-small cell lung cancer cohort. Diarrhea (61.1%), fatigue (50.4%) and hypertension (46.9%) were the most frequent treatment-emergent adverse events. Most treatment-emergent adverse events were mild-to-moderate in severity. The study closed before the planned number of patients were enrolled in all cohorts. Conclusion: Sitravatinib had a manageable safety profile with modest signals of clinical activity in patients with molecularly selected solid tumors.Clinical trial registration: www.clinicaltrials.gov identifier is NCT02219711.

Sitravatinib (MGCD516) is an oral inhibitor of several closely related oncogenic tyrosine kinase receptors that include VEGFR-2 (vascular endothelial growth factor receptor-2), AXL, and MET (mesenchymal-epithelial transition). The safety and antitumor activity of sitravatinib are reported in patients from two histologic cohorts (anti-angiogenesis-refractory clear cell renal cell carcinoma [RCC] and castrate-resistant prostate cancer [CRPC] with bone metastases) who participated in a Phase 1/1b study. The patients were enrolled using a 3-stage design that was based on observed objective responses. Objective response rate (ORR) was the primary endpoint. Duration of response, progression-free survival (PFS), overall survival (OS), and safety were also assessed. Overall, 48 patients (RCC n = 38, CRPC n = 10) received ≥ 1 dose of sitravatinib. Both cohorts were heavily pretreated (median number of prior systemic therapies: RCC cohort 3, CRPC cohort 6). In the RCC cohort, ORR was 25.9%, P = 0.015 (null hypothesis [ORR ≤ 10%] was rejected). Responses were durable (median duration 13.2 months). Median PFS was 9.5 months and median OS was 30.0 months. No objective responses were seen in the CRPC cohort; median PFS and OS were 5.8 months and 10.1 months, respectively. Across both cohorts, diarrhea (72.9%), fatigue (54.2%), and hypertension (52.1%) were the most frequent all-cause treatment-emergent adverse events (TEAEs). Diarrhea and vomiting (both, 6.3%) were the most frequent serious TEAEs considered related to study treatment. Sitravatinib demonstrated an acceptable safety profile and promising clinical activity in patients with clear cell RCC refractory to prior angiogenesis inhibitor therapy. Strong indicators for clinical activity were not seen in patients with CRPC and bone metastases. Clinical trial registration:ClinicalTrials.gov NCT02219711., Competing Interests: Declarations. Ethics approval: The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines, defined by the International Council for Harmonisation. The study was approved by the institutional review board at each participating site. Competing interests: Shubham Pant: research support to institution for Elicio, Amal Therapeutics, Janssen, Arcus, Astellas, Biontech, Boehringer Ingelheim, Mirati Therapeutics, Bristol Myers Squibb, 4D Pharma, Framewave, ImmunoMET, Ipsen, Lilly, NGM Pharmaceuticals, Novartis; Pfizer, Xencor, Immuneering, and Zymeworks; and consulting/advisory fees from USWorldmeds, Alligator Bioscience, Boehringer Ingelheim, Novartis, BPGBio, Ipsen, AstraZeneca, Janssen, Jazz Pharmaceuticals, AskGene Pharma, Nihon Medi-Physics Co, Ltd, and Zymeworks. Byoung Chul Cho: research funding from Cyrus, Abbvie, AstraZeneca, Dizal Pharma, Bayer, Blueprint Medicines, Bridgebio Therapeutics, CHA Bundang Medical Center, Champions Oncology, CJ Blossom Park, Dong-A ST, Genexine, GI-Cell, CJ Bioscience, GI-ImmuneOncia, Hanmi, Abion, Illumina, Innovation, Interpark Bio Convergence Corp, Janssen, JINTSbio, Kanaph Therapeutics, LG Chem, Boehringer Ingelheim, Lilly, MOGAM Institute, MSD, Novartis, Nuvalent, Ono, Oscotec, Regeneron, Therapex, Vertical Bio AG, Oncternal, and Yuhan; consulting fees from AstraZeneca, Janssen, BeiGene, Novartis, Boehringer-Ingelheim, CJ, CureLogen, Cyrus Therapeutics, Abion, Eli Lilly, GI-Cell, Guardant, Hanmi, Bristol Myers Squibb, HK Inno-N, Imnewrun Biosciences Inc., Medpacto, MSD, Ono, Onegene Biotechnology, Pfizer, RandBio, Takeda, Blueprint Medicines, and Yuhan; honoraria from Chinese Thoracic Oncology Society, International Association for the Study of Lung Cancer, American Society of Clinical Oncology, European Society of Medical Oncology, Guardant, Pfizer, AstraZeneca, Korean Cancer Study Group, Korean Society of Medical Onoclogy, Korean Society of Thyroid, Head and Neck Surgery, MSD, Novartis, Roche, and Korean Cancer Association; advisory board roles for Cyrus Therapeutics, Bridgebio Therapeutics, Guardant Health, J INTS BIO, Gilead, KANAPH Therapeutic Inc, Oscotec Inc, Amgen, and Therapex; royalties from Crown Bioscience, Bio GmbH, Champions Oncology, PearlRiver, Imagen, and Roche; member of the board of directors for J INTS BIO; founder of DAAN Biotherapeutics; and stock ownership for Cyrus Therapeutics, Gencurix Inc, Interpark Bio Convergence Corp., J INTS BIO, KANAPH Therapeutics, Bridgebio Therapeutics, and TheraCanVac Inc. Christos E. Kyriakopoulos: research support from Bristol Myers Squibb, Merck, AstraZeneca, Gilead Sciences, Pionyr Immunopharma, Incyte Corporation, and Sanofi-Aventis; participation in Advisory Boards for Janssen, AVEO Pharmaceuticals, Exelixis, and Sanofi-Aventis; stock for EPIC Systems Inc. Alexander Spira: honoraria from Bristol Myers Squibb, Amgen, Novartis, Bayer, CytomX Therapeutics, Janssen Oncology, Merck, AstraZeneca/MedImmune, and Takeda; consulting/advisory roles for Incyte,Amgen, Blueprint Medicines, Array Biopharma, AstraZeneca/MedImmune, Black Diamond Therapeutics, Janssen Research & Development; Bristol Myers Squibb, Mersana, Daiichi Sankyo, Sanofi, Gritstone Oncology, Jazz Pharmaceuticals, Lilly, Merck, Mirati Therapeutics, Novartis, Regeneron, and Takeda; research funding from Abbvie, Bristol Myers Squibb, ADCT, Alkermes, Arch Therapeutics, Arrivent Biopharma, Astellas Pharma, AstraZeneca/MedImmune, Ignyta, Astex Pharmaceuticals, Plexxikon,Black Diamond Therapeutics, Gritstone Oncology, Blueprint Medicines, Boehringer Ingelheim, Roche, CytomX Therapeutics, Revolution Medicines, Amgen, Daiichi Sankyo, Incyte, LAM Therapeutics, Loxo, Macrogenics, Medikine, Mersana, Mirati Therapeutics, Synthekine, Janssen Oncology, Nalo Therapeutics, Novartis, Regeneron, Rubius, Scorpion Therapeutics, and Takeda; leadership role for NEXT Oncology (Virginia); and stock ownership for Eli Lilly Nizar Tannir: clinical grants from Exelixis, Bristol Myers Squibb, Nektar Pharmaceuticals, Calithera Biosciences, and Novartis; honoraria for consultancy/advisory roles from Exelixis, AstraZeneca, Eisai, Intellisphere, Merck, Nektar Therapeutics, Bristol Myers Squibb, and Oncorena; and stock or stock options for Biocryst Pharmaceuticals, Merck, Surface Oncology, Amgen, Vanguard Healthcare, Johnson & Johnson/Janssen, and SPDR S&P Pharmaceuticals. Theresa L. Werner: research support to institution for Mersana Therapeutics, Abbvie, GSK-Tesaro, Acrivon, BluePrint Medicines, Clovis Oncology, Genmab, Repare Therapeutics, AstraZeneca, and Roche Genetech; and honoraria for advisory roles from Mersana Therapeutics Xiaohong Yan: employment, stock ownership and support for meeting attendance for Mirati Therapeutics Inc. Saskia Neuteboom: prior employment and stock ownership for Mirati Therapeutics Inc. Richard Chao: employment, stock ownership and support for meeting attendance for Mirati Therapeutics Inc. Sanjay Goel: research support to institution from Mirati Therapeutics Inc., (© 2024. The Author(s).)

Sitravatinib (MGCD516), a spectrum-selective receptor tyrosine kinase inhibitor targeting TAM (TYRO3, AXL, MERTK) and split kinase family receptors, has demonstrated preclinical anti-tumor activity and modulation of tumor microenvironment. This first-in-human phase 1/1b study included sitravatinib dose exploration and anti-tumor activity evaluation in selected patients with advanced solid tumors. Primary objectives included assessment of safety, pharmacokinetics and clinical activity of sitravatinib. Secondary objectives included identifying doses for further investigation and exploring molecular markers for patient selection. In phase 1, 32 patients received 10-200 mg, while phase 1b dose expansion comprised 161 patients (150 mg n = 99, 120 mg n = 62). Maximum tolerated dose was determined as 150 mg daily. Dose-limiting toxicity was reported in 4/28 evaluable phase 1 patients (three at 200 mg, one at 80 mg). In phase 1b, 120 mg was defined as the recommended dose due to tolerability. Treatment-related adverse events (TRAEs) were experienced by 174/193 patients (90.2%); grade ≥ 3 TRAEs in 103 patients (53.4%). Most common TRAEs were diarrhea, fatigue, hypertension and nausea; TRAEs led to treatment discontinuation in 26 patients (13.5%) and death in one patient. Sitravatinib was steadily absorbed and declined from plasma with a terminal elimination half-life of 42.1-51.5 h following oral administration. Overall objective response rate was 11.8% in phase 1b, 13.2% in patients with non-small cell lung cancer (NSCLC) and 4.2% in patients with NSCLC with prior checkpoint inhibitor experience. Sitravatinib demonstrated manageable safety and modest clinical activity in solid tumors. NCT02219711 (first posted August 14, 2014)., (© 2022. The Author(s).)

KPU-105 (4), a potent anti-microtubule agent that contains a benzophenone was derived from the diketopiperazine-type vascular disrupting agent (VDA) plinabulin 3, which displays colchicine-like tubulin depolymerization activity. To develop derivatives with more potent anti-microtubule and cytotoxic activities, we further modified the benzophenone moiety of 4. Accordingly, we obtained a 4-fluorobenzophenone derivative 16j that inhibited tumor cell growth in vitro with a subnanomolar IC(50) value against HT-29 cells (IC(50)=0.5 nM). Next, the effect of 16j on mitotic spindles was evaluated in HeLa cells. Treatment with 3nM of 16j partially disrupted the interphase microtubule network. By contrast, treatment with the same concentration of CA-4 barely affected the microtubule network, indicating that 16j exhibited more potent anti-mitotic effects than did CA-4., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Plinabulin (11, NPI-2358) is a potent microtubule-targeting agent derived from the natural diketopiperazine "phenylahistin" (1) with a colchicine-like tubulin depolymerization activity. Compound 11 was recently developed as VDA and is now under phase II clinical trials as an anticancer drug. To develop more potent antimicrotubule and cytotoxic derivatives based on the didehydro-DKP skeleton, we performed further modification on the tert-butyl or phenyl groups of 11, and evaluated their cytotoxic and tubulin-binding activities. In the SAR study, we developed more potent derivatives 33 with 2,5-difluorophenyl and 50 with a benzophenone in place of the phenyl group. The anti-HuVEC activity of 33 and 50 exhibited a lowest effective concentration of 2 and 1 nM for microtubule depolymerization, respectively. The values of 33 and 50 were 5 and 10 times more potent than that of CA-4, respectively. These derivatives could be a valuable second-generation derivative with both vascular disrupting and cytotoxic activities.

The proteasome has emerged as an important clinically relevant target for the treatment of hematologic malignancies. Since the Food and Drug Administration approved the first-in-class proteasome inhibitor bortezomib (Velcade) for the treatment of relapsed/refractory multiple myeloma (MM) and mantle cell lymphoma, it has become clear that new inhibitors are needed that have a better therapeutic ratio, can overcome inherent and acquired bortezomib resistance and exhibit broader anti-cancer activities. Marizomib (NPI-0052; salinosporamide A) is a structurally and pharmacologically unique β-lactone-γ-lactam proteasome inhibitor that may fulfill these unmet needs. The potent and sustained inhibition of all three proteolytic activities of the proteasome by marizomib has inspired extensive preclinical evaluation in a variety of hematologic and solid tumor models, where it is efficacious as a single agent and in combination with biologics, chemotherapeutics and targeted therapeutic agents. Specifically, marizomib has been evaluated in models for multiple myeloma, mantle cell lymphoma, Waldenstrom's macroglobulinemia, chronic and acute lymphocytic leukemia, as well as glioma, colorectal and pancreatic cancer models, and has exhibited synergistic activities in tumor models in combination with bortezomib, the immunomodulatory agent lenalidomide (Revlimid), and various histone deacetylase inhibitors. These and other studies provided the framework for ongoing clinical trials in patients with MM, lymphomas, leukemias and solid tumors, including those who have failed bortezomib treatment, as well as in patients with diagnoses where other proteasome inhibitors have not demonstrated significant efficacy. This review captures the remarkable translational studies and contributions from many collaborators that have advanced marizomib from seabed to bench to bedside.

NPI-2358 (1) is a potent antimicrotubule agent that was developed from a natural diketopiperazine, phenylahistin, which is currently in Phase I clinical trials as an anticancer drug. To understand the precise recognition mechanism of tubulin by this agent, we focused on its potent derivative, KPU-244 (2), which has been modified with a photoreactive benzophenone structure, and biotin-tagged KPU-244 derivatives (3 and 4), which were designed and synthesized for tubulin photoaffinity labeling. Introduction of the biotin structure at the p'-position of the benzophenone ring in 2 exhibited reduced, but significant biological activities with tubulin binding, tubulin depolymerization and cytotoxicity in comparison to the parent KPU-244. Therefore, tubulin photoaffinity labeling studies of biotin-derivatives 3 and 4 were performed by using Western blotting analysis after photoirradiation with 365 nm UV light. The results indicated that tubulin was covalently labeled by these biotin-tagged photoprobes. The labeling of compound 4 was competitively inhibited by the addition of diketopiperazine 1 or colchicine, and weakly inhibited by the addition of vinblastine. The results suggest that photoaffinity probe 4 specifically recognizes tubulin at the same binding site as anticancer drug candidate 1, and this leads to the disruption of microtubules. Probe 4 serves well as a useful chemical probe for potent antimicrotubule diketopiperazines, much like phenylahistin, and it also competes for the colchicine-binding site.

ELAC2 is a novel candidate cancer susceptibility gene located on chromosome 17p: Carriers of mutations in ELAC2 display a higher risk of developing prostate cancer. Overexpression of ELAC2 in tumor cells causes a delay in G2-M progression characterized by accumulation of cyclin B levels. Consistent with a function in mitosis, further biochemical analysis revealed that ELAC2 physically interacts with the gamma-tubulin complex. This is the first biologic insight into the function of this new putative cancer susceptibility gene, providing clues of how perturbation of ELAC2 might promote tumorigenesis through irregular cell division., (Copyright 2003 Wiley-Liss, Inc.)

In vitro experiments have demonstrated intercellular trafficking of the VP22 tegument protein of herpes simplex virus type 1 from infected cells to neighboring cells, which internalize VP22 and transport it to the nucleus. VP22 also can mediate intercellular transport of fusion proteins, providing a strategy for increasing the distribution of therapeutic proteins in gene therapy. Intercellular trafficking of the p53 tumor suppressor protein was demonstrated in vitro using a plasmid expressing full-length p53 fused in-frame to full-length VP22. The p53-VP22 chimeric protein induced apoptosis both in transfected tumor cells and in neighboring cells, resulting in a widespread cytotoxic effect. To evaluate the anti-tumor activity of p53-VP22 in vivo, we constructed recombinant adenoviruses expressing either wild-type p53 (FTCB) or a p53-VP22 fusion protein (FVCB) and compared their effects in p53-resistant tumor cells. In vitro, treatment of tumor cells with FVCB resulted in enhanced p53-specific apoptosis compared to treatment with equivalent doses of FTCB. However, in normal cells there was no difference in the dose-related cytotoxicity of FVCB compared to that of FTCB. In vivo, treatment of established tumors with FVCB was more effective than equivalent doses of FTCB. The dose-response curve to FVCB was flatter than that to FTCB; maximal antitumor responses could be achieved using FVCB at doses 1 log lower than those obtained with FTCB. Increased antitumor efficacy was correlated with increased distribution of p53 protein in FVCB-treated tumors. This study is the first demonstration that VP22 can enhance the in vivo distribution of therapeutic proteins and improve efficacy in gene therapy.

TP53 is the most commonly altered tumor-suppressor gene in cancer and is currently being tested in Phase II/III gene replacement trials. Many tumors contain wild-type TP53 sequence with elevated MDM2 protein levels, targeting p53 for degradation. These tumors are more refractory to treatment with exogenous wild-type p53. Here we generate a recombinant adenovirus expressing a p53 variant, rAd-p53 (d 13-19), that is deleted for the amino acid sequence necessary for MDM2 binding (amino acids 13-19). We compared the apoptotic activity of rAd-p53 (d 13-19) with that of a recombinant adenovirus expressing wild-type p53 (rAd-p53) in cell lines that differ in endogenous p53 status. rAd-p53 (d 13-19) caused higher levels of apoptosis in p53 wild-type tumor lines compared with wild-type p53 treatment, as measured by annexin V-FITC staining. In p53-altered tumor lines, rAd-p53 (d 13-19) showed apoptotic activity similar to that seen with wild-type p53 treatment. In normal cells, no increase in cytopathicity was detected with rAd-p53 (d 13-19) compared with wild-type p53 treatment. This variant protein displayed synergy with chemotherapeutic agents to inhibit proliferation of ovarian and breast cell lines. The p53 variant showed greater antitumor activity in an established p53 wild-type tumor compared with treatment with wild-type p53. The p53 variant represents a means of expanding TP53 gene therapy to tumors that are resistant to p53 treatment due to the cellular responses to wild-type p53.

A large fraction of pediatric pre-B acute lymphoblastoid leukemias (ALL) consistently contain a t(1;19) chromosomal translocation. The t(1;19) translocation results in the production of a chimeric transcription factor containing the N-terminal transactivation domain of E2A fused to the C-terminal DNA-binding homeodomain of Pbx1. Here, we show that the E2A-Pbx1 fusion protein activates the expression of a novel WNT gene, WNT-16. WNT-16 normally is expressed in peripheral lymphoid organs such as spleen, appendix, and lymph nodes, but not in bone marrow. In contrast, high levels of WNT-16 transcripts are present in bone marrow and cell lines derived from pre-B ALL patients carrying the E2A-Pbx1 hybrid gene. Inhibition of E2A-Pbx1 expression leads to a significant decrease in WNT-16 mRNA levels, suggesting that WNT-16 is a downstream target of E2A-Pbx1. Three putative WNT receptors, FZ-2, FZ-3, and FZ-5, are expressed in cells of the B lineage, including pre-B ALL cells aberrantly expressing WNT-16. We propose that a WNT-16-mediated autocrine growth mechanism contributes to the development of t(1;19) pre-B ALL.

Genetic alterations in the MMAC1 tumor suppressor gene (also referred to as PTEN or TEP1) occur in several types of human cancers including glioblastoma. Growth suppression induced by overexpression of MMAC1 in cells with mutant MMAC1 alleles is thought to be mediated by the inhibition of signaling through the phosphatidylinositol 3-kinase pathway. However, the exact biochemical mechanisms by which MMAC1 exerts its growth-inhibitory effects are still unknown. Here we report that recombinant adenovirus-mediated overexpression of MMAC1 in three different MMAC1-mutant glioblastoma cell lines blocked progression from G0/G1 to S phase of the cell cycle. Cell cycle arrest correlated with the recruitment of the cyclin-dependent kinase (CDK) inhibitor, p27Kip1, to cyclin E immunocomplexes, which resulted in a reduction in CDK2 kinase activities and a decrease in levels of endogenous phosphorylated retinoblastoma protein. CDK4 kinase activities were unaffected, as were the levels of the CDK inhibitor p21Cip1 present in cyclin E immunocomplexes. Therefore, overexpression of MMAC1 via adenovirus-mediated gene transfer suppresses tumor cell growth through cell cycle inhibitory mechanisms, and as such, represents a potential therapeutic approach to treating glioblastomas.

A novel DNA-binding activity, designated CBF, has been identified in nuclear extracts from tobacco leaf, stem and root tissue. CBF interacts specifically with a 30 bp promoter fragment, referred to as cyt-1, of the Agrobacterium tumefaciens T-DNA cytokinin (T-cyt) gene. The T-cyt promoter, although of bacterial origin is active in planta and the 30 bp cyt-1 element is located within a region that is essential for T-cyt promotor activity in leaf, stem and root cells of tobacco plants. Gel retardation assays using different synthetic oligonucleotides and methylation interference experiments pinpointed the binding site of CBF to a GC-rich sequence ATGCCCCACA within the cyt-1 element. Site-directed mutagenesis of the CBF binding site within the T-cyt promoter by using PCR resulted in an almost complete loss of T-cyt promoter activity in transgenic tobacco plants. In a gain-of-function experiment a hexamer of cyt-1 was shown to be able to confer leaf, stem and root expression when fused upstream of a TATA box containing -55 derivative of the T-cyt promoter. A mutant cyt-1 hexamer, defective in CBF binding, did not show activity above background levels. These results indicate that binding of CBF to the cyt-1 element is required for cyt-1 directed gene expression, suggesting that CBF might act as a transcriptional activator. Apart from the ASF-1 binding site of the CaMV 35S promoter, which is also present in the T-DNA nopaline and octopine synthase genes, the cyt-1 element is the only other identified element reported until now that in combination with a TATA box is sufficient to drive gene expression in multiple tobacco tissue types.

The promoter region of the Agrobacterium tumefaciens T-cyt gene was fused to a beta-glucuronidase (gusA) reporter gene and introduced into tobacco plants. Detection of gusA expression in transgenic F1 progeny revealed that the T-cyt promoter is active in many, if not all, cell types in leaves, stems and roots of fully developed plants. Developmental stage-dependent promoter activity was observed in seedlings. Analysis of 5'-deleted promoter fragments showed that sequences located between positions -185 and -139 with respect to the T-cyt translational start codon are essential for T-cyt promoter activity in transfected tobacco protoplasts as well as in transformed tobacco plants.

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified by the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.