ABSTRACT
CpG oligodeoxynucleotides are toll-like receptor 9 agonists capable of inducing potent pro-inflammatory immune responses. Although CpG oligodeoxynucleotides have shown promising antitumor effects, their systemic activity can trigger immune-related toxicity, limiting therapeutic application. We previously identified glatiramer acetate (GA), a cationic polypeptide approved for the treatment of relapsing-remitting multiple sclerosis, as an intratumoral delivery agent capable of complexing with CpG, thereby pinning it to the injection site and limiting systemic exposure. Here, we investigated whether the combination of CpG or GA-CpG polyplexes and intraperitoneal anti-PD-1 therapy would result in synergistic efficacy in AT84 and CT26 murine syngeneic models of head and neck and colon cancers, respectively. In both AT84 and CT26 tumor models, intratumoral CpG or GA-CpG treatment similarly suppressed tumor growth, but the efficacy was not amplified with anti-PD-1. Nevertheless, combination treatment increased cytotoxic T cell, helper T cell, and natural killer cell infiltration into AT84 tumors. Surprisingly, the combination of intratumoral GA and intraperitoneal anti-PD-1 treatment resulted in elevated systemic GM-CSF and IL-2 cytokine levels and demonstrated synergistic antitumor effects in the CT26 mouse tumor model. Moreover, tumors that responded most significantly to anti-PD-1 plus GA treatment showed increased markers of infiltration of CD4+ T cells and natural killer cells. Combinations of intratumoral GA or GA-CpG polyplexes with anti-PD-1 treatment warrant further investigation as combination cancer immunotherapy strategies.
Subject(s)
Immunotherapy , Neoplasms , Mice , Animals , Glatiramer Acetate/therapeutic use , Immunotherapy/methods , Oligodeoxyribonucleotides , Adjuvants, Immunologic/therapeutic use , Adjuvants, Immunologic/pharmacology , Neoplasms/drug therapy , Cell Line, TumorABSTRACT
A rapid and effective method to identify disease-specific antibodies from clinical patients is important for understanding autoimmune diseases and for the development of effective disease therapies. In neuromyelitis optica (NMO), the identification of antibodies targeting the aquaporin-4 (AQP4) membrane protein traditionally involves the labor-intensive and time-consuming process of single B-cell sorting, followed by antibody cloning, expression, purification, and analysis for anti-AQP4 activity. To accelerate patient-specific antibody discovery, we compared two unique approaches for screening anti-AQP4 antibodies from yeast antibody surface display libraries. Our first approach, cell-based biopanning, has strong advantages for its cell-based display of native membrane-bound AQP4 antigens and is inexpensive and simple to perform. Our second approach, FACS screening using solubilized AQP4 antigens, permits real-time population analysis and precision sorting for specific antibody binding parameters. We found that both cell-based biopanning and FACS screening were effective for the enrichment of AQP4-binding clones. These screening techniques will enable library-scale functional interrogation of large natively paired antibody libraries for comprehensive analysis of anti-AQP4 antibodies in clinical samples and for robust therapeutic discovery campaigns.
ABSTRACT
Multiple sclerosis is complex and heterogeneous. Better tools are needed to be able to monitor this disease among individuals, but blood-based biomarkers are often too rare to profile. In this work, we developed antigen-specific biomaterials to replicate the central nervous system niche where multiple sclerosis biomarkers are amplified. We incorporated mouse brain homogenate into a microporous gelatin methacrylate network. Homogenate-containing biomaterials differentially stimulated cells and led to the marked amplification of disease-relevant, antigen-specific B cells. These results demonstrate that biomaterials containing primary tissue homogenate retain antigen specificity and may be a useful tool for decoding human autoimmunity.
Subject(s)
Antigens/metabolism , Biocompatible Materials/chemistry , Brain/metabolism , Animals , Antigens/chemistry , Autoimmunity , B-Lymphocytes/cytology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , B7-2 Antigen/metabolism , Encephalomyelitis, Autoimmune, Experimental/metabolism , Encephalomyelitis, Autoimmune, Experimental/pathology , Gelatin/chemistry , Mice , Myelin Proteolipid Protein/chemistry , Myelin Proteolipid Protein/immunology , Myelin Proteolipid Protein/metabolism , Peptide Fragments/chemistry , Peptide Fragments/immunology , Peptide Fragments/metabolism , Spleen/cytology , Spleen/metabolismABSTRACT
Fc fusions are a growing class of drugs comprising an antibody Fc domain covalently linked to a protein or peptide and can pose manufacturing challenges. In this study we evaluated three synthetic approaches to generate Fc fusions, using Fc-insulin as a model drug candidate. Engineered human IgG1 was digested with HRV3C to produce an Fc fragment with a C-terminal sortase tag (Fc-LPETGGH6). The synthesis of Fc-insulin2 from Fc-LPETGGH6 was evaluated with direct sortase-mediated ligation (SML) and two chemoenzymatic strategies. Direct SML was performed with triglycine-insulin, and chemoenzymatic strategies used to SML fuse either triglycine-azide or triglycine-DBCO prior to linking insulin with copper-catalyzed or strain-promoted azidealkyne cycloaddition. Reaction conditions were optimized by evaluating reagent concentrations, relative equivalents, temperature, and time. Direct SML provided the most effective reaction yields, converting 60-70% of Fc-LPETGGH6 to Fc-insulin2, whereas our optimized chemoenzymatic synthesis converted 30-40% of Fc-LPETGGH6 to Fc-insulin2. Here we show that SML is a practical and efficient method to synthesize Fc fusions and provide an optimized pathway for fusion drug synthesis.
ABSTRACT
Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.
Subject(s)
Antigens/chemistry , Antigens/immunology , B-Lymphocytes/immunology , Fluorescent Dyes/chemistry , Polymerization , Animals , Click Chemistry , Diabetes Mellitus, Type 1/immunology , Humans , Mice , Multiple Sclerosis/immunologyABSTRACT
Many autoimmune therapies focus on immune suppression to reduce symptom severity and halt disease progression; however, currently approved treatments lack specificity for the autoantigen and rely on more global immune suppression. Multivalent antigen arrays can disarm pathogenic autoimmune B cell populations that specifically recognize the antigen of interest via their B cell receptor (BCR). Disarmament may be achieved by BCR engagement, cross-linking, and sustained receptor occupancy as a result of multivalent, high avidity BCR binding. To engage and explore this mechanism, a tetramer display of the encephalogenic proteolipid peptide (PLP139-151), referred to as 4-arm PLP139-151, was synthesized by copper-catalyzed azide-alkyne cycloaddition chemistry. Subcutaneous administration of 4-arm PLP139-151 completely ameliorated symptoms of paralysis in a mouse model of multiple sclerosis known as experimental autoimmune encephalomyelitis. Competitive binding of 4-arm PLP139-151 to PLP139-151-specific IgG in the mouse serum demonstrated the enhanced avidity associated with the multivalent array compared to the free peptide. Furthermore, key PLP139-151-reactive B cells were depleted following 4-arm PLP139-151 treatment, resulting in significant reduction of proinflammatory cytokines. Together, these data demonstrate the potential of 4-arm PLP139-151 to silence autoreactive B cell populations and limit the downstream activation of effector cells.
Subject(s)
Autoantigens/administration & dosage , B-Lymphocytes/immunology , Encephalomyelitis, Autoimmune, Experimental/therapy , Immune Tolerance , Immunotherapy/methods , Multiple Sclerosis/therapy , Myelin Proteolipid Protein/administration & dosage , Peptide Fragments/administration & dosage , Administration, Topical , Animals , Autoantigens/blood , Autoantigens/immunology , Encephalomyelitis, Autoimmune, Experimental/blood , Encephalomyelitis, Autoimmune, Experimental/immunology , Female , Immunoglobulin G/blood , Mice , Multiple Sclerosis/blood , Multiple Sclerosis/immunology , Myelin Proteolipid Protein/blood , Myelin Proteolipid Protein/immunology , Paralysis/blood , Paralysis/immunology , Paralysis/therapy , Peptide Fragments/blood , Peptide Fragments/immunology , Receptors, Antigen, B-Cell/immunology , Treatment OutcomeABSTRACT
Cancer therapies aim to kill tumor cells directly or engage the immune system to fight malignancy. Checkpoint inhibitors, oncolytic viruses, cell-based immunotherapies, cytokines, and adjuvants have been applied to prompt the immune system to recognize and attack cancer cells. However, systemic exposure of cancer therapies can induce unwanted adverse events. Intratumoral administration of potent therapies utilizes small amounts of drugs, in an effort to minimize systemic exposure and off-target toxicities. Here, we discuss the properties of the tumor microenvironment and transport considerations for intratumoral drug delivery. Specifically, we consider various tumor tissue factors and physicochemical factors that can affect tumor retention after intratumoral injection. We also review approved and clinical-stage intratumoral therapies and consider how the molecular and biophysical properties (e.g. size and charge) of these therapies influences intratumoral transport (e.g. tumor retention and cellular uptake). Finally, we offer a critical review and highlight several emerging approaches to promote tumor retention and limit systemic exposure of potent intratumoral therapies.
Subject(s)
Neoplasms , Oncolytic Virotherapy , Oncolytic Viruses , Pharmaceutical Preparations , Humans , Immunotherapy , Neoplasms/drug therapy , Tumor MicroenvironmentABSTRACT
Antigen-specific immunotherapies (ASIT) present compelling potential for introducing precision to the treatment of autoimmune diseases where nonspecific, global immunosuppression is currently the only treatment option. Central to ASIT design is the delivery of autoantigen, which parallels allergy desensitization approaches. Clinical success in tolerizing allergen-specific responses spans longer than a century, but autoimmune ASITs have yet to see an FDA-approved breakthrough. Allergens and autoantigens differ substantially in physicochemical properties, and these discrepancies influence the nature of their interactions with the immune system. Approved allergen-specific immunotherapies are typically administered as water soluble, neutrally charged protein fractions from 10 to 70â¯kDa. Conversely, autoantigens are native proteins that exhibit wide-ranging sizes, solubilities, and charges that render them susceptible to immunogenicity. To translate the success of allergen hyposensitization to ASIT, delivery strategies may be necessary to effectively format autoantigens, guide biodistribution, and engage appropriate immune mechanisms.
Subject(s)
Autoantigens/immunology , Drug Delivery Systems/methods , Immunotherapy/methods , Allergens/chemistry , Allergens/pharmacology , Autoimmune Diseases/physiopathology , Biological Transport/physiology , Desensitization, Immunologic/methods , Drug Administration Routes , HumansABSTRACT
Adaptive immunity plays a central role in the pathogenesis of type 1 diabetes. Among past treatment approaches, B cell ablation has yielded unmistakable therapeutic potency; however, global immunosuppression imposes unacceptable risks to a patient population consisting of children. Multivalent antigen arrays represent a compelling strategy for targeted immunosuppression by selectively engaging and inactivating autoreactive B cells. Here, we report the design and characterization of 4-arm polyethylene glycol-insulin (PEG-Ins) conjugates as multivalent arrays for autoreactive B cell engagement. First, we selectively modified human insulin at the B29 residue to retain antigenicity. Next, we conjugated the modified proteins to 20 kDa, 4-arm polyethylene glycol backbones to produce multivalent PEG-Ins constructs. Mass spectrometry, circular dichroism, and dynamic light scattering indicated that the structure of insulin was maintained in the much larger, multivalent construct. PEG-Ins conjugates demonstrated an ex vivo immunological effect in splenocytes harboring an anti-insulin B cell receptor (VH125SD) by inactivating B cells and promoting an anergic phenotype that was downregulated in B cell receptor expression (CD79b), and PEG-Ins conjugates did not mobilize calcium upon B cell receptor stimulation. These data support the further study of PEG-Ins conjugates in animal models of type 1 diabetes.
ABSTRACT
A synthetic peptide, K-PLP, consisting of 11-unit poly-lysine (K11) linked via polyethylene glycol (PEG) to proteolipid protein epitope (PLP) was synthesized, characterized, and evaluated for efficacy in ameliorating experimental autoimmune encephalomyelitis (EAE) induced by PLP. K-PLP was designed to mimic the cationic nature of the relapsing-remitting multiple sclerosis treatment, glatiramer acetate (GA). With a pI of ~10, GA is able to form visible aggregates at the site of injection via electrostatic interactions with the anionic extracellular matrix. Aggregation further facilitates the retention of GA at the site of injection and draining lymph nodes, which may contribute to its mechanism of action. K-PLP with a pI of ~11, was found to form visible aggregates in the presence of glycosaminoglycans and persist at the injection site and draining lymph nodes in vivo, similar to GA. Additionally, EAE mice treated with K-PLP showed significant inhibition of clinical symptoms compared to free poly-lysine and to PLP, which are the components of K-PLP. The ability of the poly-lysine motif to retain PLP at the injection site, which increased the local exposure of PLP to immune cells may be an important factor affecting drug efficacy.
Subject(s)
Autoantigens/administration & dosage , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Glatiramer Acetate/administration & dosage , Immunosuppressive Agents/administration & dosage , Molecular Mimicry , Peptides/administration & dosage , Animals , Autoantigens/chemistry , Autoantigens/metabolism , Cells, Cultured , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Female , Glatiramer Acetate/metabolism , Immunosuppressive Agents/metabolism , Injections, Subcutaneous , Lymph Nodes/drug effects , Lymph Nodes/immunology , Lymph Nodes/metabolism , Mice , Peptides/chemical synthesis , Peptides/metabolism , Protein Aggregates , Spleen/drug effects , Spleen/immunology , Spleen/metabolism , Tissue DistributionABSTRACT
Relapsing-remitting patterns of many autoimmune diseases such as multiple sclerosis (MS) are perpetuated by a recurring circuit of adaptive immune cells that amplify in secondary lymphoid organs (SLOs) and traffic to compartments where antigen is abundant to elicit damage. Some of the most effective immunotherapies impede the migration of immune cells through this circuit, however, broadly suppressing immune cell migration can introduce life-threatening risks for patients. We developed antigen-specific immune decoys (ASIDs) to mimic tissues targeted in autoimmunity and selectively intercept autoimmune cells to preserve host tissue. Using Experimental Autoimmune Encephalomyelitis (EAE) as a model, we conjugated autoantigen PLP139-151 to a microporous collagen scaffold. By subcutaneously implanting ASIDs after induction but prior to the onset of symptoms, mice were protected from paralysis. ASID implants were rich with autoimmune cells, however, reactivity to cognate antigen was substantially diminished and apoptosis was prevalent. ASID-implanted mice consistently exhibited engorged spleens when disease normally peaked. In addition, splenocyte antigen-presenting cells were highly activated in response to PLP rechallenge, but CD3+ and CD19â¯+â¯effector subsets were significantly decreased, suggesting exhaustion. ASID-implanted mice never developed EAE relapse symptoms even though the ASID material had long since degraded, suggesting exhausted autoimmune cells did not recover functionality. Together, data suggested ASIDs were able to sequester and exhaust immune cells in an antigen-specific fashion, thus offering a compelling approach to inhibit the migration circuit underlying autoimmunity.
Subject(s)
Autoimmune Diseases/immunology , Autoimmunity/physiology , Animals , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Autoantigens/immunology , Autoimmune Diseases/therapy , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/therapy , Female , Immune Tolerance , Immunotherapy , MiceABSTRACT
Antigen valency plays a fundamental role in directing the nature of an immune response to be stimulatory or tolerogenic. Soluble antigen arrays (SAgAs) are an antigen-specific immunotherapy that combats autoimmunity through the multivalent display of autoantigen. Although mechanistic studies have shown SAgAs to induce T- and B-cell anergy, the effect of SAgA valency has never been experimentally tested. Here, SAgAs of discrete antigen valencies were synthesized by click chemistry and evaluated for acute B-cell signaling inhibition as well as downstream immunomodulatory effects in splenocytes. Initial studies using the Raji B-cell line demonstrated SAgA valency dictated the extent of calcium flux. Lower valency constructs elicited the largest reductions in B-cell activation. In splenocytes from mice with experimental autoimmune encephalomyelitis, the same valency-dependent effects were evident in the downregulation of the costimulatory marker CD86. The reduction of calcium flux observed in Raji B-cells correlated strongly with downregulation in splenocyte CD86 expression after 72 h. Here, a thorough analysis of SAgA antigenic valency illustrates that low, but not monovalent, presentation of autoantigen was ideal for eliciting the most potent immunomodulatory effects.
Subject(s)
Autoantigens/chemistry , B-Lymphocytes/immunology , Immunomodulation , Spleen/cytology , Animals , Autoantigens/immunology , B7-2 Antigen/immunology , Cell Line, Tumor , Cells, Cultured , Click Chemistry/methods , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/therapy , Humans , Immunoassay/methods , Mice , Protein Array Analysis/methods , Spleen/immunologyABSTRACT
Contemporary approaches to treating autoimmune diseases like multiple sclerosis broadly modulate the immune system and leave patients susceptible to severe adverse effects. Antigen-specific immunotherapies (ASIT) offer a unique opportunity to selectively suppress autoreactive cell populations but have suffered from marginal efficacy even when employing traditional adjuvants to improve delivery. The development of immunologically active antigen delivery vehicles could potentially increase the clinical success of antigen-specific immunotherapies. An emulsion of the antioxidant tocopherol delivering an epitope of proteolipid protein autoantigen (PLP139-151) yielded significant efficacy in mice with experimental autoimmune encephalomyelitis (EAE). In vitro studies indicated tocopherol emulsions reduced oxidative stress in antigen-presenting cells. Ex vivo analysis revealed that tocopherol emulsions shifted cytokine responses in EAE splenocytes. In addition, IgG responses against PLP139-151 were increased in mice treated with tocopherol emulsions delivering the antigen, suggesting a possible skew in immunity. Overall, tocopherol emulsions provide a functional delivery vehicle for ASIT capable of ameliorating autoimmunity in a murine model.
Subject(s)
Autoantigens/therapeutic use , Emulsions/chemistry , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Tocopherols/chemistry , Tocopherols/therapeutic use , Animals , Autoantigens/administration & dosage , Cytokines/metabolism , Female , Immune Tolerance/drug effects , Immunotherapy/methods , Mice , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/pathogenicity , Spleen/cytologyABSTRACT
Autoreactive lymphocytes that escape central immune tolerance may be silenced via an endogenous peripheral tolerance mechanism known as anergy. Antigen-specific therapies capable of inducing anergy may restore patients with autoimmune diseases to a healthy phenotype while avoiding deleterious side effects associated with global immunosuppression. Inducing anergy in B cells may be a particularly potent intervention, as B cells can contribute to autoimmune diseases through multiple mechanisms and offer the potential for direct antigen-specific targeting through the B cell receptor (BCR). Our previous results suggested autoreactive B cells may be silenced by multivalent 'soluble antigen arrays' (SAgAs), which are polymer conjugates displaying multiple copies of autoantigen with or without a secondary peptide that blocks intracellular cell-adhesion molecule-1 (ICAM-1). Here, key therapeutic molecular properties of SAgAs were identified and linked to the immunological mechanism through comprehensive cellular and in vivo analyses. We determined non-hydrolyzable 'cSAgAs' displaying multivalent 'click'-conjugated antigen more potently suppressed experimental autoimmune encephalomyelitis (EAE) compared to hydrolyzable SAgAs capable of releasing conjugated antigen. cSAgAs restored a healthy phenotype in disease-specific antigen presenting cells (APCs) by inducing an anergic response in B cells and a subset of B cells called autoimmune-associated B cells (ABCs) that act as potent APCs in autoimmune disease. Accompanied by a cytokine response skewed towards a Th2/regulatory phenotype, this generated an environment of autoantigenic tolerance. By identifying key therapeutic molecular properties and an immunological mechanism that drives SAgA efficacy, this work guides the design of antigen-specific immunotherapies capable of inducing anergy.
Subject(s)
Autoantigens/genetics , B-Lymphocyte Subsets/drug effects , Clonal Anergy/drug effects , Encephalomyelitis, Autoimmune, Experimental/therapy , Immunoconjugates/pharmacology , Immunotherapy/methods , Peptide Fragments/pharmacology , Animals , Autoantigens/immunology , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/pathology , Click Chemistry , Dendritic Cells/immunology , Dendritic Cells/pathology , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , Female , Hydrolysis , Immunoconjugates/chemistry , Injections, Subcutaneous , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/immunology , Mice , Myelin Proteolipid Protein/administration & dosage , Peptide Fragments/administration & dosage , Peptide Fragments/chemical synthesis , Peptide Fragments/immunology , Protein Array Analysis , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, B-Cell/immunology , Spleen/immunology , Spleen/pathology , Th2 Cells/immunology , Th2 Cells/pathologyABSTRACT
Current therapies for autoimmune diseases focus on treating the symptoms rather than the underlying disease cause. A major setback in improving current therapeutics for autoimmunity is the lack of antigen specificity. Successful antigen-specific immunotherapy (ASIT) would allow for improved treatment of autoimmune diseases. In this work, dexamethasone was co-delivered with autoantigen (PLP) in vivo to create effective ASIT for the treatment of experimental autoimmune encephalomyelitis (EAE). Using an emulsion of incomplete Freund's adjuvant (IFA) as a co-delivery vehicle, it was discovered that the controlled release of autoantigen was important for the suppression of clinical disease symptoms. Analysis of the immune response via cytokines revealed that dexamethasone was important for shifting the immune response away from inflammation. Co-delivery of both autoantigen and dexamethasone increased B-cell populations and antibody production, signifying an increased humoral immune response. Overall, this data indicated that the co-delivery of PLP and dexamethasone with a water-in-oil emulsion is effective in treating a murine autoimmune model.
Subject(s)
Anti-Inflammatory Agents/administration & dosage , Autoantigens/administration & dosage , Dexamethasone/administration & dosage , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Freund's Adjuvant/administration & dosage , Immunologic Factors/administration & dosage , Lipids/administration & dosage , Myelin Proteolipid Protein/administration & dosage , Peptide Fragments/administration & dosage , Animals , Antibody Formation , B-Lymphocytes/immunology , Cytokines/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Female , Mice , Spleen/cytologyABSTRACT
BACKGROUND: Weight loss benefits of multi-ingredient supplements in conjunction with a low-calorie, high-protein diet in young women are unknown. Therefore, the purpose of this study was to investigate the effects of a three-week low-calorie diet with and without supplementation on body composition. METHODS: Thirty-seven recreationally-trained women (n = 37; age = 27.1 ± 4.2; height = 165.1 ± 6.4; weight = 68.5 ± 10.1; BMI = 25.1 ± 3.4) completed one of the following three-week interventions: no change in diet (CON); a high-protein, low-calorie diet supplemented with a thermogenic, conjugated linoleic acid (CLA), a protein gel, and a multi-vitamin (SUP); or the high-protein diet with isocaloric placebo supplements (PLA). Before and after the three-week intervention, body weight, %Fat via dual X-ray absorptiometry (DXA), segmental fat mass via DXA, %Fat via skinfolds, and skinfold thicknesses at seven sites were measured. RESULTS: SUP and PLA significantly decreased body weight (SUP: PRE, 70.47 ± 8.01 kg to POST, 67.51 ± 8.10 kg; PLA: PRE, 67.88 ± 12.28 kg vs. POST, 66.38 ± 11.94 kg; p ≤ 0.05) with a greater (p ≤ 0.05) decrease in SUP than PLA or CON. SUP and PLA significantly decreased %Fat according to DXA (SUP: PRE, 34.98 ± 7.05% to POST, 32.99 ± 6.89%; PLA: PRE, 34.22 ± 6.36% vs. POST, 32.69 ± 5.84%; p ≤ 0.05), whereas only SUP significantly decreased %Fat according to skinfolds (SUP: PRE, 27.40 ± 4.09% to POST, 24.08 ± 4.31%; p ≤ 0.05). SUP significantly (p ≤ 0.05) decreased thicknesses at five skinfolds (chest, waist, hip, subscapular, and tricep) compared to PLA, but not at two skinfolds (axilla and thigh). CONCLUSIONS: The addition of a thermogenic, CLA, protein, and a multi-vitamin to a three-week low-calorie diet improved weight loss, total fat loss and subcutaneous fat loss, compared to diet alone.