Lawrence J. Thomas

Vaccine-Induced Antibodies Inhibit CETP Activity In Vivo and Reduce Aortic Lesions in a Rabbit Model of Atherosclerosis

Using a vaccine approach, we immunized New Zealand White rabbits with a peptide containing a region of cholesteryl ester transfer protein (CETP) known to be required for neutral lipid transfer function. These rabbits had significantly reduced plasma CETP activity and an altered lipoprotein profile. In a cholesterol-fed rabbit model of atherosclerosis, the fraction of plasma cholesterol in HDL was 42% higher and the fraction of plasma cholesterol in LDL was 24% lower in the CETP-vaccinated group than in the control-vaccinated group. Moreover, the percentage of the aorta surface exhibiting atherosclerotic lesion was 39.6% smaller in the CETP-vaccinated rabbits than in controls. The data reported here demonstrate that CETP activity can be reduced in vivo by vaccination with a peptide derived from CETP and support the concept that inhibition of CETP activity in vivo can be antiatherogenic. In addition, these studies suggest that vaccination against a self-antigen is a viable therapeutic strategy for disease management.

Soluble CR1 Therapy Improves Complement Regulation in C3 Glomerulopathy

Dense deposit disease (DDD) and C3 glomerulonephritis (C3GN) are widely recognized subtypes of C3 glomerulopathy. These ultra-rare renal diseases are characterized by fluid-phase dysregulation of the alternative complement pathway that leads to deposition of complement proteins in the renal glomerulus. Disease triggers are unknown and because targeted treatments are lacking, progress to end stage renal failure is a common final outcome. We studied soluble CR1, a potent regulator of complement activity, to test whether it restores complement regulation in C3 glomerulopathy. In vitro studies using sera from patients with DDD showed that soluble CR1 prevents dysregulation of the alternative pathway C3 convertase, even in the presence of C3 nephritic factors. In mice deficient in complement factor H and transgenic for human CR1, soluble CR1 therapy stopped alternative pathway activation, resulting in normalization of serum C3 levels and clearance of iC3b from glomerular basement membranes. Short-term use of soluble CR1 in a pediatric patient with end stage renal failure demonstrated its safety and ability to normalize activity of the terminal complement pathway. Overall, these data indicate that soluble CR1 re-establishes regulation of the alternative complement pathway and provide support for a limited trial to evaluate soluble CR1 as a treatment for DDD and C3GN.

Agonist Anti-Human CD27 Monoclonal Antibody Induces T Cell Activation and Tumor Immunity in Human CD27–Transgenic Mice

The CD70/CD27 pathway plays a significant role in the control of immunity and tolerance, and previous studies demonstrated that targeting murine CD27 (mCD27) with agonist mAbs can mediate antitumor efficacy. We sought to exploit the potential of this pathway for immunotherapy by developing 1F5, a fully human IgG1 mAb to human CD27 (hCD27) with agonist activity. We developed transgenic mice expressing hCD27 under control of its native promoter for in vivo testing of the Ab. The expression and regulation of hCD27 in hCD27-transgenic (hCD27-Tg) mice were consistent with the understood biology of CD27 in humans. In vitro, 1F5 effectively induced proliferation and cytokine production from hCD27-Tg–derived T cells when combined with TCR stimulation. Administration of 1F5 to hCD27-Tg mice enhanced Ag-specific CD8+ T cell responses to protein vaccination comparably to an agonist anti-mCD27 mAb. In syngeneic mouse tumor models, 1F5 showed potent antitumor efficacy and induction of protective immunity, which was dependent on CD4+ and CD8+ T cells. The requirement of FcR engagement for the agonistic and antitumor activities of 1F5 was demonstrated using an aglycosylated version of the 1F5 mAb. These data with regard to the targeting of hCD27 are consistent with previous reports on targeting mCD27 and provide a rationale for the clinical development of the 1F5 mAb, for which studies in advanced cancer patients have been initiated under the name CDX-1127.

Development of a Human Monoclonal Antibody for Potential Therapy of CD27-Expressing Lymphoma and Leukemia

Purpose: The TNF receptor superfamily member CD27 is best known for its important role in T-cell immunity but is also recognized as a cell-surface marker on a number of B- and T-cell malignancies. In this article, we describe a novel human monoclonal antibody (mAb) specific for CD27 with properties that suggest a potential utility against malignancies that express CD27. Experimental Design: The fully human mAb 1F5 was generated using human Ig transgenic mice and characterized by analytical and functional assays in vitro. Severe combined immunodeficient (SCID) mice inoculated with human CD27-expressing lymphoma cells were administered 1F5 to investigate direct antitumor effects. A pilot study of 1F5 was conducted in non-human primates to assess toxicity. Results: 1F5 binds with high affinity and specificity to human and macaque CD27 and competes with ligand binding. 1F5 activates T cells only in combination with T-cell receptor stimulation and does not induce proliferation of primary CD27-expressing tumor cells. 1F5 significantly enhanced the survival of SCID mice bearing Raji or Daudi tumors, which may be mediated through direct effector mechanisms such as antibody-dependent cellular cytotoxicity. Importantly, administration of up to 10 mg/kg of 1F5 to cynomolgus monkeys was well tolerated without evidence of significant toxicity or depletion of circulating lymphocytes. Conclusions: Collectively, the data suggest that the human mAb 1F5, which has recently entered clinical development under the name CDX-1127, may provide direct antitumor activity against CD27-expressing lymphoma or leukemia, independent of its potential to enhance immunity through its agonistic properties. Clin Cancer Res; 18(14); 3812–21. ©2012 AACR.

Targeting human CD27 with an agonist antibody stimulates T-cell activation and antitumor immunity.

CD27 is an important co-stimulatory receptor of T cells that can potentially be exploited for immunotherapy. We developed a human IgG1 antibody that targets human CD27, and demonstrated its immunostimulatory and antineoplastic activity in various preclinical models. Currently, the antibody (1F5, CDX-1127) is being tested in patients affected by advanced malignancies.

Co-administration of a CpG adjuvant (VaxImmuneTM, CPG 7909) with CETP vaccines increased immunogenicity in rabbits and mice

Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that facilitates the transfer of neutral lipids and phospholipids between lipoproteins and contributes to the regulation of the plasma concentration of high density lipoprotein cholesterol (HDL-C). Vaccines have been developed that elicit antibodies that bind to and reduce the lipid transfer function of CETP as a way to increase the plasma concentration of HDL-C and prevent or treat atherosclerosis. This study assessed the immunogenicity of two vaccine peptides. The first, CETi-1, is a dimerized synthetic peptide, including residues 461-476 of human CETP and residues 830-843 of tetanus toxoid, TT(830-843). The second, PADRE-CETP, is a monomeric peptide, in which a PADRE T cell epitope (aK-Cha-VAAWTLKAa) replaces the TT(830-843) T cell epitope of CETi-1. Both peptides were formulated with aluminum-containing adjuvants (Alhydrogel®), and tested in mice and rabbits with or without the co-administration of the investigational TLR9 agonist VaxImmuneTM (CPG 7909). In both mice and rabbits, the vaccine peptide utilizing the PADRE T cell epitope elicited stronger anti-CETP antibody responses than the CETi-1 vaccine. Also, co-administration of VaxImmune enhanced the anti-CETP antibody responses to both vaccines. Isotype analysis of the murine anti-CETP antibody response to both vaccines demonstrated a switch from IgG1 to IgG2a upon co-administration of VaxImmune. We conclude that 1) the PADRE T cell epitope is more potent than the TT(830-843) epitope in providing help for the anti-CETP antibody response; and 2) co-administration of VaxImmune with either vaccine increased immunogenicity as measured by antibody response.

Vaccination against anthrax

Methods are disclosed for immunizing a mammal against B. anthracis using a composition of pure recombinant Protective Antigen (rPA), optionally in combination with truncated Lethal Factor polypeptide (LFn). Formulations of the pure rPA immunogen have little or no reactogenicity and therefore may be administered to a mammalian subject in very high doses of 50 μg to 1000 μg or more rPA, which is at least four times the amount of PA included per dose in conventional anthrax vaccines. Preferred immunogenic compositions are free of adjuvant and other undesired components, further enhancing the effectiveness and safety of the compositions. Methods for preparing the immunogenic compositions and for purifying rPA and LFn polypeptides also are disclosed.

CD27-Mediated Regulatory T Cell Depletion and Effector T Cell Costimulation Both Contribute to Antitumor Efficacy

CD27, a member of the TNFR superfamily, is constitutively expressed in most T cells and plays crucial roles in T cell effector functions. The costimulation and antitumor activity of CD27 agonistic Abs have been well documented in mouse models. Clinical testing of a human IgG1 anti-CD27 Ab, varlilumab (clone 1F5), is ongoing in cancer patients. In this study, we set out to further understand CD27 as an immunomodulatory target and to address the mechanism of antitumor efficacy using different IgG isotypes of 1F5 in human CD27-transgenic mice. 1F5mIgG1, the only isotype engaging inhibitory FcγRIIB expressed in B cells, elicited the most potent and broad immune response, but terminal differentiation, exhaustion, and apoptosis in the activated effector T cells were inevitable. Accordingly, this isotype was the most effective in eradicating BCL1 lymphoma but had limited efficacy in s.c. tumors. Conversely, 1F5mIgG2a, which interacts with cells expressing activating FcγRs, led to moderate immune activation, as well as to prominent reduction in the number and suppressive activity of regulatory T cells. These combined mechanisms imparted potent antitumor activity to 1F5mIgG2a, particularly against the s.c. tumors. 1F5hIgG1, varlilumab, showed balanced agonistic activity that was prominent at lower doses and depleting activity that was greater at higher doses. 1F5hIgG1 had good antitumor activity in all tumor models tested. Thus, both agonist and depleting properties contribute to the antitumor efficacy of CD27-targeted immunotherapy, and modulation of these activities in patients may be achieved by varying the dose and regimen.

Characterization of N‐linked oligosaccharides bearing sialyl Lewis x moieties on an alternatively glycosylated form of soluble complement receptor type 1 (sCR1)

We sought to produce a complement inhibitory protein possessing oligosaccharides specifically modified to contain the sialyl Lewis x (sLex) moiety. This modified glycoprotein could combine anti‐complement activity with the ability to inhibit selectin‐mediated interactions and concentrate this activity to sites of activated endothelium where selectins are upregulated. Soluble complement receptor type 1 (sCR1), previously shown to be effective in inhibiting the complement cascade, was produced in a cell line capable of adding fucose to N‐linked oligosaccharides in the α1‐3 linkage, which is necessary for sLex glycosylation. The glycoprotein purified from these cells was designated sCR1sLex, and may prove to be more effective than sCR1 in some clinical applications. Detailed analysis and characterization of sCR1sLex was performed to confirm that the N‐linked oligosaccharides possessed sLex moieties and also to determine the extent of sLex glycosylation. The glycoproteins were characterized by oligosaccharide profiling, sequencing, linkage analysis and quantified by differential enzymic digestion, using fluorophore‐assisted carbohydrate electrophoresis. The major glycans were identified as biantennary oligosaccharides (including sialylated and non‐core fucosylated glycans). The linkages of sialic acid and the branched fucose were analysed by digestion with linkage‐specific enzymes and subsequent separation by electrophoresis. All data were consistent with the presence of sLex moieties on the N‐linked oligosaccharides of sCR1sLex. sCR1sLex is a prime example of a recombinant protein expressed with oligosaccharides engineered for a specific biological function, and produced using a commercially viable method.

Development of a Novel Antibody-drug Conjugate for the Potential Treatment of Ovarian, Lung and Renal Cell Carcinoma Expressing TIM-1

T-cell immunoglobulin and mucin domain 1 (TIM-1) is a type I transmembrane protein that was originally described as kidney injury molecule 1 (KIM-1) due to its elevated expression in kidney and urine after renal injury. TIM-1 expression is also upregulated in several human cancers, most notably in renal and ovarian carcinomas, but has very restricted expression in healthy tissues, thus representing a promising target for antibody-mediated therapy. To this end, we have developed a fully human monoclonal IgG1 antibody specific for the extracellular domain of TIM-1. This antibody was shown to bind purified recombinant chimeric TIM-1-Fc protein and TIM-1 expressed on a variety of transformed cell lines, including Caki-1 (human renal clear cell carcinoma), IGROV-1 (human ovarian adenocarcinoma), and A549 (human lung carcinoma). Internalization studies using confocal microscopy revealed the antibody was rapidly internalized by cells in vitro, and internalization was confirmed by quantitative imaging flow cytometry. An antibody–drug conjugate (ADC) was produced with the anti-TIM-1 antibody covalently linked to the potent cytotoxin, monomethyl auristatin E (MMAE), and designated CDX-014. The ADC was shown to exhibit in vitro cytostatic or cytotoxic activity against a variety of TIM-1–expressing cell lines, but not on TIM-1–negative cell lines. Using the Caki-1, IGROV-1, and A549 xenograft mouse models, CDX-014 showed significant antitumor activity in a clinically relevant dose range. Safety evaluation in nonhuman primates has demonstrated a good profile and led to the initiation of clinical studies of CDX-014 in renal cell carcinoma and potentially other TIM-1–expressing tumors. Mol Cancer Ther; 15(12); 2946–54. ©2016 AACR.