Publications by authors named "Vanitha Ramakrishnan"

17 Publications

  • Page 1 of 1

TriTACs, a Novel Class of T-Cell-Engaging Protein Constructs Designed for the Treatment of Solid Tumors.

Mol Cancer Ther 2021 01 17;20(1):109-120. Epub 2020 Nov 17.

Harpoon Therapeutics, South San Francisco, California.

T cells have a unique capability to eliminate cancer cells and fight malignancies. Cancer cells have adopted multiple immune evasion mechanisms aimed at inhibiting T cells. Dramatically improved patient outcomes have been achieved with therapies genetically reprogramming T cells, blocking T-cell inhibition by cancer cells, or transiently connecting T cells with cancer cells for redirected lysis. This last modality is based on antibody constructs that bind a surface antigen on cancer cells and an invariant component of the T-cell receptor. Although high response rates were observed with T-cell engagers specific for CD19, CD20, or BCMA in patients with hematologic cancers, the treatment of solid tumors has been less successful. Here, we developed and characterized a novel T-cell engager format, called TriTAC (for Trispecific T-cell Activating Construct). TriTACs are engineered with features to improve patient safety and solid tumor activity, including high stability, small size, flexible linkers, long serum half-life, and highly specific and potent redirected lysis. The present study establishes the structure/activity relationship of TriTACs and describes the development of HPN424, a PSMA- (FOLH1-) targeting TriTAC in clinical development for patients with metastatic castration-resistant prostate cancer.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0061DOI Listing
January 2021

Zanubrutinib monotherapy for patients with treatment naïve chronic lymphocytic leukemia and 17p deletion.

Haematologica 2020 Oct 13;Online ahead of print. Epub 2020 Oct 13.

Dana-Farber Cancer Institute, Boston, MA.

Patients with chronic lymphocytic leukemia or small lymphocytic lymphoma whose tumors carry deletion of chromosome 17p13.1 [del(17p)] have an unfavorable prognosis and respond poorly to standard chemoimmunotherapy. Zanubrutinib is a selective next-generation Bruton tyrosine kinase inhibitor. We evaluated the safety and efficacy of zanubrutinib 160 mg twice daily in treatment-naïve patients with del(17p) disease enrolled in a dedicated, nonrandomized cohort (Arm C) of the phase 3 SEQUOIA trial. A total of 109 patients (median age, 70 years; range, 42 - 86) with centrally confirmed del(17p) were enrolled and treated. After a median of 18.2 months (range, 5.0 - 26.3), seven patients had discontinued study treatment due to progressive disease, four due to an adverse event, and one due to withdrawal of consent. The overall response rate was 94.5% with 3.7% of patients achieving complete response with or without incomplete hematologic recovery. The estimated 18-month progression-free survival rate was 88.6% (95% CI, 79.0 - 94.0) and the estimated 18-month overall survival rate was 95.1% (95% CI, 88.4 - 98.0). Most common all-grade adverse events included contusion (20.2%), upper respiratory tract infection (19.3%), neutropenia/neutrophil count decreased (17.4%), and diarrhea (16.5%). Grade ≥ 3 adverse events were reported in 53 patients (48.6%), most commonly neutropenia (12.9%) and pneumonia (3.7%). An adverse event of atrial fibrillation was reported in three patients (2.8%). Zanubrutinib was active and well tolerated in this large, prospectively enrolled treatment cohort of previously untreated patients with del(17p) chronic lymphocytic leukemia/small lymphocytic lymphoma. This trial was registered at ClinicalTrials.gov as #NCT03336333.
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http://dx.doi.org/10.3324/haematol.2020.259432DOI Listing
October 2020

Phase I study of the anti-FcRH5 antibody-drug conjugate DFRF4539A in relapsed or refractory multiple myeloma.

Blood Cancer J 2019 02 4;9(2):17. Epub 2019 Feb 4.

Sarah Cannon Research Institute, Nashville, TN, USA.

FcRH5 is a cell surface marker enriched on malignant plasma cells when compared to other hematologic malignancies and normal tissues. DFRF4539A is an anti-FcRH5 antibody-drug conjugated to monomethyl auristatin E (MMAE), a potent anti-mitotic agent. This phase I study assessed safety, tolerability, maximum tolerated dose (MTD), anti-tumor activity, and pharmacokinetics of DFRF4539A in patients with relapsed/refractory multiple myeloma. DFRF4539A was administered at 0.3-2.4 mg/kg every 3 weeks or 0.8-1.1 mg/kg weekly as a single-agent by intravenous infusion to 39 patients. Exposure of total antibody and antibody-conjugate-MMAE analytes was linear across the doses tested. There were 37 (95%) adverse events (AEs), 8 (21%) serious AEs, and 15 (39%) AEs ≥ grade 3. Anemia (n = 10, 26%) was the most common AE considered related to DFRF4539A. Two cases of grade 3 acute renal failure were attributed to DFRF4539A. There were no deaths; the MTD was not reached. DFRF4539A demonstrated limited activity in patients at the doses tested with 2 (5%) partial response, 1 (3%) minimal response, 18 (46%) stable disease, and 16 (41%) progressive disease. FcRH5 was confirmed to be expressed and occupied by antibody post-treatment and thus remains a valid myeloma target. Nevertheless, this MMAE-based antibody-drug-conjugate targeting FcRH5 was unsuccessful for myeloma.
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http://dx.doi.org/10.1038/s41408-019-0178-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362066PMC
February 2019

Potential mechanisms for thrombocytopenia development with trastuzumab emtansine (T-DM1).

Clin Cancer Res 2015 Jan 4;21(1):123-33. Epub 2014 Nov 4.

Department of Project Management and Operations, Genentech, Inc, South San Francisco, California.

Purpose: Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) comprising the cytotoxic agent DM1 conjugated to trastuzumab with a stable linker. Thrombocytopenia was the dose-limiting toxicity in the phase I study, and grade ≥3 thrombocytopenia occurred in up to 13% of patients receiving T-DM1 in phase III studies. We investigated the mechanism of T-DM1-induced thrombocytopenia.

Experimental Design: The effect of T-DM1 on platelet function was measured by aggregometry, and by flow cytometry to detect the markers of activation. The effect of T-DM1 on differentiation and maturation of megakaryocytes (MK) from human hematopoietic stem cells was assessed by flow cytometry and microscopy. Binding, uptake, and catabolism of T-DM1 in MKs, were assessed by various techniques including fluorescence microscopy, scintigraphy to detect T-[H(3)]-DM1 and (125)I-T-DM1, and mass spectrometry. The role of FcγRIIa was assessed using blocking antibodies and mutant constructs of trastuzumab that do not bind FcγR.

Results: T-DM1 had no direct effect on platelet activation and aggregation, but it did markedly inhibit MK differentiation via a cytotoxic effect. Inhibition occurred with DM1-containing ADCs but not with trastuzumab demonstrating a role for DM1. MKs internalized these ADCs in a HER2-independent, FcγRIIa-dependent manner, resulting in intracellular release of DM1. Binding and internalization of T-DM1 diminished as MKs matured; however, prolonged exposure of mature MKs to T-DM1 resulted in a disrupted cytoskeletal structure.

Conclusions: These data support the hypothesis that T-DM1-induced thrombocytopenia is mediated in large part by DM1-induced impairment of MK differentiation, with a less pronounced effect on mature MKs.
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http://dx.doi.org/10.1158/1078-0432.CCR-14-2093DOI Listing
January 2015

Biomarker analyses from a placebo-controlled phase II study evaluating erlotinib±onartuzumab in advanced non-small cell lung cancer: MET expression levels are predictive of patient benefit.

Clin Cancer Res 2014 Sep 31;20(17):4488-98. Epub 2014 Mar 31.

Genentech Inc., South San Francisco;

Purpose: In a recent phase II study of onartuzumab (MetMAb), patients whose non-small cell lung cancer (NSCLC) tissue scored as positive for MET protein by immunohistochemistry (IHC) experienced a significant benefit with onartuzumab plus erlotinib (O+E) versus erlotinib. We describe development and validation of a standardized MET IHC assay and, retrospectively, evaluate multiple biomarkers as predictors of patient benefit.

Experimental Design: Biomarkers related to MET and/or EGF receptor (EGFR) signaling were measured by IHC, FISH, quantitative reverse transcription PCR, mutation detection techniques, and ELISA.

Results: A positive correlation between IHC, Western blotting, and MET mRNA expression was observed in NSCLC cell lines/tissues. An IHC scoring system of MET expression taking proportional and intensity-based thresholds into consideration was applied in an analysis of the phase II study and resulted in the best differentiation of outcomes. Further analyses revealed a nonsignificant overall survival (OS) improvement with O+E in patients with high MET copy number (mean≥5 copies/cell by FISH); however, benefit was maintained in "MET IHC-positive"/MET FISH-negative patients (HR, 0.37; P=0.01). MET, EGFR, amphiregulin, epiregulin, or HGF mRNA expression did not predict a significant benefit with onartuzumab; a nonsignificant OS improvement was observed in patients with high tumor MET mRNA levels (HR, 0.59; P=0.23). Patients with low baseline plasma hepatocyte growth factor (HGF) exhibited an HR for OS of 0.519 (P=0.09) in favor of onartuzumab treatment.

Conclusions: MET IHC remains the most robust predictor of OS and progression-free survival benefit from O+E relative to all examined exploratory markers.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-1836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4504679PMC
September 2014

A Phase Ib study evaluating MNRP1685A, a fully human anti-NRP1 monoclonal antibody, in combination with bevacizumab and paclitaxel in patients with advanced solid tumors.

Cancer Chemother Pharmacol 2014 May 17;73(5):951-60. Epub 2014 Mar 17.

South Texas Accelerated Research Therapeutics, 4383 Medical Drive, San Antonio, TX, 78229, USA,

Purpose: MNRP1685A is a human monoclonal antibody that blocks binding of vascular endothelial growth factor (VEGF), VEGF-B, and placental growth factor 2 to neuropilin-1 resulting in vessel immaturity and VEGF dependency. The safety of combining MNRP1685A with bevacizumab, with or without paclitaxel, was examined.

Methods: Patients with advanced solid tumors received escalating doses of MNRP1685A (7.5, 15, 24, and 36 mg/kg) with bevacizumab 15 mg/kg every 3 weeks in Arm A (n = 14). Arm B (n = 10) dosing consisted of MNRP1685A (12 and 16 mg/kg) with bevacizumab 10 mg/kg (every 2 weeks) and paclitaxel 90 mg/m(2) (weekly, 3 of 4 weeks). Objectives were to determine safety, pharmacokinetics, pharmacodynamics, and the maximum tolerated dose of MNRP1685A.

Results: Infusion reactions (88 %) and transient thrombocytopenia (67 %) represent the most frequent study drug-related adverse events (AEs). Drug-related Grade 2 or 3 proteinuria occurred in 13 patients (54 %). Additional study drug-related AEs occurring in >20 % of patients included neutropenia, alopecia, dysphonia, fatigue, and nausea. Neutropenia occurred only in Arm B. Grade ≥3 study drug-related AEs in ≥3 patients included neutropenia (Arm B), proteinuria, and thrombocytopenia. Two confirmed and three unconfirmed partial responses were observed.

Conclusions: The safety profiles were consistent with the single-agent profiles of all study drugs. However, a higher than expected rate of clinically significant proteinuria was observed that does not support further testing of MNRP1685A in combination with bevacizumab.
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http://dx.doi.org/10.1007/s00280-014-2426-8DOI Listing
May 2014

Evaluation and clinical analyses of downstream targets of the Akt inhibitor GDC-0068.

Clin Cancer Res 2013 Dec 18;19(24):6976-86. Epub 2013 Oct 18.

Authors' Affiliations: Oncology Biomarker Development, Portfolio Management and Operations, Translational Oncology, Biostatistics, and Exploratory Clinical Development, Genentech Inc., DNA Way, South San Francisco, California; Experimental Therapeutics, Molecular Pathology, Breast Cancer and Melanoma, Gastrointestinal and Endocrine Tumors Groups, and Medical Oncology Service, Vall d'Hebron Institute of Oncology, Barcelona; Department of Hematology and Medical Oncology, Institute of Health Research INCLIVA, University of Valencia, Valencia, Spain; and Human Oncology and Pathogenesis Program and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York.

Purpose: The oncogenic PI3K/Akt/mTOR pathway is an attractive therapeutic target in cancer. However, it is unknown whether the pathway blockade required for tumor growth inhibition is clinically achievable. Therefore, we conducted pharmacodynamic studies with GDC-0068, an ATP competitive, selective Akt1/2/3 inhibitor, in preclinical models and in patients treated with this compound.

Experimental Design: We used a reverse phase protein array (RPPA) platform to identify a biomarker set indicative of Akt inhibition in cell lines and human-tumor xenografts, and correlated the degree of pathway inhibition with antitumor activity. Akt pathway activity was measured using this biomarker set in pre- and post-dose tumor biopsies from patients treated with GDC-0068 in the dose escalation clinical trial.

Results: The set of biomarkers of Akt inhibition is composed of 10 phosphoproteins, including Akt and PRAS40, and is modulated in a dose-dependent fashion, both in vitro and in vivo. In human-tumor xenografts, this dose dependency significantly correlated with tumor growth inhibition. Tumor biopsies from patients treated with GDC-0068 at clinically achievable doses attained a degree of biomarker inhibition that correlated with tumor growth inhibition in preclinical models. In these clinical samples, compensatory feedback activation of ERK and HER3 was observed, consistent with preclinical observations.

Conclusion: This study identified a set of biomarkers of Akt inhibition that can be used in the clinical setting to assess target engagement. Here, it was used to show that robust Akt inhibition in tumors from patients treated with GDC-0068 is achievable, supporting the clinical development of this compound in defined patient populations.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-0978DOI Listing
December 2013

DCDT2980S, an anti-CD22-monomethyl auristatin E antibody-drug conjugate, is a potential treatment for non-Hodgkin lymphoma.

Mol Cancer Ther 2013 Jul 18;12(7):1255-65. Epub 2013 Apr 18.

Genentech Research and Early Development, 1 DNA Way, South San Francisco, CA 94080, USA.

Antibody-drug conjugates (ADC), potent cytotoxic drugs linked to antibodies via chemical linkers, allow specific targeting of drugs to neoplastic cells. We have used this technology to develop the ADC DCDT2980S that targets CD22, an antigen with expression limited to B cells and the vast majority of non-Hodgkin lymphomas (NHL). DCDT2980S consists of a humanized anti-CD22 monoclonal IgG1 antibody with a potent microtubule-disrupting agent, monomethyl auristatin E (MMAE), linked to the reduced cysteines of the antibody via a protease cleavable linker, maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB). We describe the efficacy, safety, and pharmacokinetics of DCDT2980S in animal models to assess its potential as a therapeutic for the treatment of B-cell malignancies. We did not find a strong correlation between in vitro or in vivo efficacy and CD22 surface expression, nor a correlation of sensitivity to free drug and in vitro potency. We show that DCDT2980S was capable of inducing complete tumor regression in xenograft mouse models of NHL and can be more effective than rituximab plus combination chemotherapy at drug exposures that were well tolerated in cynomolgus monkeys. These results suggest that DCDT2980S has an efficacy, safety, and pharmacokinetics profile that support potential treatment of NHL.
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http://dx.doi.org/10.1158/1535-7163.MCT-12-1173DOI Listing
July 2013

Investigational antibody-drug conjugates for hematological malignancies.

Expert Opin Investig Drugs 2011 Jan 11;20(1):75-85. Epub 2010 Dec 11.

Genentech, Inc., Department of Translational Oncology, 1 DNA way, Mail Stop 72A, South San Francisco 94080, CA, USA.

Importance Of The Field: Antibody-drug conjugates (ADCs) consist of potent cytotoxic drugs linked to antibodies via chemical linkers. ADCs facilitate the specific targeting of drugs to neoplastic cells. This technology is showing efficacy with manageable toxicity for the treatment of hematological malignancies.

Areas Covered In This Review: ADCs for the treatment of hematological malignancies are in pre-clinical and early clinical trials. This review describes these ADCs in detail and explores the challenges of optimizing the use of this technology.

What The Reader Will Gain: The reader should understand that, although ADCs are conceptually simple, the application of this idea to practice has not been straightforward, and the challenges of developing ADCs include identifying targets with appropriate expression profiles and biology, developing successful linker chemistries, and the selection of a potent cytotoxic drug.

Take Home Message: Hematological malignancies are particularly suited to the development of ADC therapeutics as their surface proteins are well characterized, and the consequences of expression of the target in the normal tissue like the bone marrow results in manageable toxicities since, in many cases, the normal tissue can regenerate. While this technology is complex, the ADCs for hematological malignancies currently in clinical use show great promise.
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http://dx.doi.org/10.1517/13543784.2011.539557DOI Listing
January 2011

Antibodies to TWEAK receptor inhibit human tumor growth through dual mechanisms.

Clin Cancer Res 2010 Jan 12;16(2):497-508. Epub 2010 Jan 12.

Facet Biotech, Redwood City, California 94063, USA.

Purpose: Targeted therapeutics have significantly changed the outcome for patients diagnosed with cancer. Still, effective therapeutic intervention does not exist for many cancers and much remains to be done. The objective of this study was to identify novel genes that potentially regulate tumor growth, to target these gene products with monoclonal antibodies, and to examine the therapeutic potential of these antibodies.

Experimental Design: Using cDNA microarray analysis, we identified genes overexpressed in several solid malignancies. We generated a mouse monoclonal antibody, 19.2.1, and its humanized counterpart, PDL192, to one such target, TweakR (TWEAK receptor, Fn14, TNFRSF12A, CD266), and characterized the antitumor activities in vitro and in mouse xenograft models.

Results: Both 19.2.1 (mouse IgG2a) and PDL192 (human IgG1), like TWEAK, the natural ligand of TweakR, inhibited the growth of several TweakR-expressing cancer cell lines in anchorage-dependent and anchorage-independent assays in vitro. Both antibodies showed significant antitumor activity in multiple mouse xenograft models. PDL192 and 19.2.1 also induced antibody-dependent cellular cytotoxicity (ADCC) of cancer cell lines in vitro. A chimeric version of 19.2.1 containing the mouse IgG1 Fc region (19.2.1 x G1) exhibited significantly less ADCC than 19.2.1. However, 19.2. 1x G1 showed differential activity in vivo, with activity equivalent to 19.2.1 in one model, but significantly less efficacy than 19.2.1 in a second model. These results indicate that PDL192 and 19.2.1 mediate their antitumor effects by signaling through TweakR, resulting in reduced tumor cell proliferation, and by ADCC.
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http://dx.doi.org/10.1158/1078-0432.CCR-09-1929DOI Listing
January 2010

Loss of E-cadherin promotes ovarian cancer metastasis via alpha 5-integrin, which is a therapeutic target.

Cancer Res 2008 Apr;68(7):2329-39

Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, University of Chicago, Chicago, IL 60637, USA.

E-cadherin loss is frequently associated with ovarian cancer metastasis. Given that adhesion to the abdominal peritoneum is the first step in ovarian cancer dissemination, we reasoned that down-regulation of E-cadherin would affect expression of cell matrix adhesion receptors. We show here that inhibition of E-cadherin in ovarian cancer cells causes up-regulation of alpha(5)-integrin protein expression and transcription. When E-cadherin was blocked, RMUG-S ovarian cancer cells were able to attach and invade more efficiently. This greater efficiency could, in turn, be inhibited both in vitro and in vivo with an alpha(5)beta(1)-integrin-blocking antibody. When E-cadherin is silenced, alpha(5)-integrin is up-regulated through activation of an epidermal growth factor receptor/FAK/Erk1-mitogen-activated protein kinase-dependent signaling pathway and not through the canonical E-cadherin/beta-catenin signaling pathway. In SKOV-3ip1 ovarian cancer xenografts, which express high levels of alpha(5)-integrin, i.p. treatment with an alpha(5)beta(1)-integrin antibody significantly reduced tumor burden, ascites, and number of metastasis and increased survival by an average of 12 days when compared with IgG treatment (P < 0.0005). alpha(5)-Integrin expression was detected by immunohistochemistry in 107 advanced stage ovarian cancers using a tissue microarray annotated with disease-specific patient follow-up. Ten of 107 tissues (9%) had alpha(5)-integrin overexpression, and 39% had some level of alpha(5)-integrin expression. The median survival for patients with high alpha(5)-integrin levels was 26 months versus 35 months for those with low integrin expression (P < 0.05). Taken together, we have identified alpha(5)-integrin up-regulation as a molecular mechanism by which E-cadherin loss promotes tumor progression, providing an explanation for how E-cadherin loss increases metastasis. Targeting this integrin could be a promising therapy for a subset of ovarian cancer patients.
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http://dx.doi.org/10.1158/0008-5472.CAN-07-5167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665934PMC
April 2008

A function blocking anti-mouse integrin alpha5beta1 antibody inhibits angiogenesis and impedes tumor growth in vivo.

J Transl Med 2007 Nov 27;5:61. Epub 2007 Nov 27.

Department of Research, PDL Biopharma, Inc,, Fremont, CA 94555, USA.

Background: Integrins are important adhesion molecules that regulate tumor and endothelial cell survival, proliferation and migration. The integrin alpha5beta1 has been shown to play a critical role during angiogenesis. An inhibitor of this integrin, volociximab (M200), inhibits endothelial cell growth and movement in vitro, independent of the growth factor milieu, and inhibits tumor growth in vivo in the rabbit VX2 carcinoma model. Although volociximab has already been tested in open label, pilot phase II clinical trials in melanoma, pancreatic and renal cell cancer, evaluation of the mechanism of action of volociximab has been limited because this antibody does not cross-react with murine alpha5beta1, precluding its use in standard mouse xenograft models.

Methods: We generated a panel of rat-anti-mouse alpha5beta1 antibodies, with the intent of identifying an antibody that recapitulated the properties of volociximab. Hybridoma clones were screened for analogous function to volociximab, including specificity for alpha5beta1 heterodimer and blocking of integrin binding to fibronectin. A subset of antibodies that met these criteria were further characterized for their capacities to bind to mouse endothelial cells, inhibit cell migration and block angiogenesis in vitro. One antibody that encompassed all of these attributes, 339.1, was selected from this panel and tested in xenograft models.

Results: A panel of antibodies was characterized for specificity and potency. The affinity of antibody 339.1 for mouse integrin alpha5beta1 was determined to be 0.59 nM, as measured by BIAcore. This antibody does not significantly cross-react with human integrin, however 339.1 inhibits murine endothelial cell migration and tube formation and elicits cell death in these cells (EC50 = 5.3 nM). In multiple xenograft models, 339.1 inhibited the growth of established tumors by 40-60% (p < 0.05) and this inhibition correlates with a concomitant decrease in vessel density.

Conclusion: The results herein demonstrate that 339.1, like volociximab, exhibits potent anti-alpha5beta1 activity and confirms that inhibition of integrin alpha5beta1 impedes angiogenesis and slows tumor growth in vivo.
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http://dx.doi.org/10.1186/1479-5876-5-61DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2235829PMC
November 2007

Volociximab, a chimeric integrin alpha5beta1 antibody, inhibits the growth of VX2 tumors in rabbits.

Invest New Drugs 2008 Feb 1;26(1):7-12. Epub 2007 Sep 1.

PDL Biopharma, Inc., 34801 Campus Drive, Fremont, CA 94555, USA.

Angiogenesis, the process by which new blood vessels form from existing vasculature, is critical for tumor growth and invasion. Growth factors, such as VEGF, initiate signaling cascades resulting in the proliferation of resting endothelial cells. Blockade of growth factor pathways has proven effective in inhibiting angiogenesis and tumor growth in vivo. Integrins, including the integrin alpha5beta1, are also important mediators of angiogenesis and these adhesion molecules also regulate cancer cell growth and migration in vitro. Volociximab is a high affinity, function-blocking antibody against integrin alpha5beta1 that is currently in multiple Phase II oncology clinical trials. Volociximab displays potent anti-angiogenic activity in a monkey model of choroidal neovascularization. In this study, we explored the consequences of integrin alpha5beta1 blockade on tumorigenesis. Because volociximab does not cross-react with rodent alpha5beta1, the syngeneic rabbit VX2 carcinoma model was utilized as an alternative to standard mouse xenograft models for the assessment of anti-tumor activity of volociximab. Volociximab administered intravenously to rabbits bearing VX2 tumors is detectable on tumor cells and vasculature 45 min post-administration. Volociximab was found to significantly inhibit the growth of tumors growing subcutaneously or intramuscularly, despite a 20-fold lower affinity for rabbit integrin, relative to human. This effect was found to correlate with decreased blood vessel density within these tumors. These results support the use of volociximab in the intervention of malignant disease.
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http://dx.doi.org/10.1007/s10637-007-9078-zDOI Listing
February 2008

Preclinical evaluation of an anti-alpha5beta1 integrin antibody as a novel anti-angiogenic agent.

J Exp Ther Oncol 2006 ;5(4):273-86

PDL Biopharma Inc, Fremont CA 94555, USA.

Integrin alpha5beta1, the principal fibronectin receptor, is an important survival factor, playing a key role in angiogenesis. Angiogenesis is critical for tumor growth, and anti-angiogenic therapies have met clinical success. To validate the therapeutic potential of an anti-alpha5beta1 strategy, we generated volociximab (M200) a chimeric human IgG4 version of the alpha5beta1 function-blocking murine antibody IIA1; and F200, the Fab derivative. Volociximab, F200 and IIA1 showed similar activity by ELISA (EC50= 0.2nM), Biacore (Kd= 0.1-0.4nM) and inhibition of fibronectin binding (IC50= 2-3nM). The inhibitory potential of alpha5beta1 antibodies was compared to HuMV833, an anti-VEGF antibody. Both volociximab and HuMV833 inhibited HUVEC proliferation (IC50 of volociximab = 0.2-0.5nM; IC50 of HuMV833 = 45nM). However, IIA1, volociximab and F200 were also potent inhibitors of an in vitro model of angiogenesis (HUVEC tube formation assay), unlike HuMV833. Additionally, volociximab inhibited in vitro tube formation induced by VEGF and/or bFGF, suggesting a mechanism of action independent of growth factor stimulus. In fact, inhibition of alpha5beta1 function by volociximab induced apoptosis of actively proliferating, but not resting, endothelial cells. Volociximab does not cross-react with rodent alpha5beta1, therefore in vivo validation of an anti-alpha5beta1 approach was conducted in a cynomolgus model of choroidal revascularization. Volociximab and F200 were potent inhibitors of neovessel formation in this model. These data demonstrate that volociximab has therapeutic potential in diseases in which new vessel formation is a component of the pathology.
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February 2007

Preclinical validation of anti-TMEFF2-auristatin E-conjugated antibodies in the treatment of prostate cancer.

Mol Cancer Ther 2004 Aug;3(8):921-32

Protein Design Labs, Inc., 34801 Campus Drive, Fremont, CA 94555, USA.

Current treatments for advanced stage, hormone-resistant prostate cancer are largely ineffective, leading to high patient mortality and morbidity. To fulfill this unmet medical need, we used global gene expression profiling to identify new potential antibody-drug conjugate (ADC) targets that showed maximal prostate cancer-specific expression. TMEFF2, a gene encoding a plasma membrane protein with two follistatin-like domains and one epidermal growth factor-like domain, had limited normal tissue distribution and was highly overexpressed in prostate cancer. Immunohistochemistry analysis using a specific monoclonal antibody (mAb) to human TMEFF2 showed significant protein expression in 74% of primary prostate cancers and 42% of metastatic lesions from lymph nodes and bone that represented both hormone-naïve and hormone-resistant disease. To evaluate anti-TMEFF2 mAbs as potential ADCs, one mAb was conjugated to the cytotoxic agent auristatin E via a cathepsin B-sensitive valine-citrulline linker. This ADC, Pr1-vcMMAE, was used to treat male severe combined immunodeficient mice bearing xenografted LNCaP and CWR22 prostate cancers expressing TMEFF2. Doses of 3 to 10 mg/kg of this specific ADC resulted in significant and sustained tumor growth inhibition, whereas an isotype control ADC had no significant effect. Similar efficacy and specificity was shown with huPr1-vcMMAE, a humanized anti-TMEFF2 ADC. No overt in vivo toxicity was observed with either murine or human ADC, despite significant cross-reactivity of anti-TMEFF2 mAb with the murine TMEFF2 protein, implying minimal toxicity to other body tissues. These data support the further evaluation and clinical testing of huPr1-vcMMAE as a novel therapeutic for the treatment of metastatic and hormone-resistant prostate cancer.
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August 2004

E-selectin up-regulation allows for targeted drug delivery in prostate cancer.

Cancer Res 2003 Oct;63(19):6387-94

Protein Design Labs, Inc., 34801 Campus Drive, Fremont, CA 94555, USA.

We have used the Eos Hu03 GeneChip array, which represents over 92% of the transcribed human genome, to measure gene expression in a panel of normal and diseased human tissues. This analysis revealed that E-selectin mRNA is selectively overexpressed in prostate cancer epithelium, a finding that correlated strongly with E-selectin protein expression as assessed by immunohistochemistry. Antibodies against E-selectin that blocked function failed to impede cancer cell growth, suggesting that overexpression of E-selectin was not essential for cell growth. However, a novel auristatin E-based antibody drug conjugate (ADC), E-selectin antibody valine-citrulline monomethyl-auristatin E, was a potent and selective agent against E-selectin-expressing cancer cell lines in vitro, with the degree of cytotoxicity varying with surface antigen density. Interestingly, sensitivity to the ADC differed among cell lines from different tissues expressing similar amounts of E-selectin and was found to correlate with sensitivity to free auristatin E. Furthermore, E-selectin-expressing tumors grown as xenografts in severe combined immunodeficient mice were responsive to treatment with E-selectin antibody valine-citrulline monomethyl-auristatin E in vivo, with more than 85% inhibition of tumor growth observed in treated mice. These findings demonstrate that an E-selectin-targeting ADC has potential as a prostate cancer therapy and validates a genomics-based paradigm for the identification of cancer-specific antigens suitable for targeted therapy.
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October 2003

Expression of the zinc transporter ZnT4 is decreased in the progression from early prostate disease to invasive prostate cancer.

Oncogene 2003 Sep;22(38):6005-12

Cancer Research Program, Garvan Institute of Medical Research, St. Vincent's Hospital, 384 Victoria Street, Darlinghurst NSW 2010, Australia.

We have utilized oligonucleotide microarrays to identify novel genes of potential clinical and biological importance in prostate cancer. RNA from 74 prostate cancers and 164 normal body samples representing 40 different tissues were analysed using a customized Affymetrix GeneChip oligonucleotide microarray representative of over 90% of the expressed human genome. The gene for the zinc transporter ZnT4 was one of several genes that displayed significantly higher expression in prostate cancer compared to normal tissues from other organs. A polyclonal antipeptide antibody was used to demonstrate ZnT4 expression in the epithelium of all 165 elements of benign and 326 elements of localized prostate cancers examined and in nine of 10 advanced prostate cancer specimens by immunohistochemistry. Interestingly, decreased intensity of ZnT4 immunoreactivity occurred in the progression from benign to invasive localized prostate cancer and to metastatic disease. Immunofluorescence analysis and surface biotinylation studies of cells expressing ZnT4 localised the protein to intracellular vesicles and to the plasma membrane. These findings are consistent with a role for ZnT4 in vesicular transport of zinc to the cell membrane and potentially in efflux of zinc in the prostate.
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http://dx.doi.org/10.1038/sj.onc.1206797DOI Listing
September 2003