Whole blood apheresis

Abstract

A method is presented for performing apheresis of mammals, including humans, that does not require separation of blood into plasma or any other fractions. Referred to herein as whole blood apheresis, this advance allows apheresis to be performed more quickly and efficiently with less stress for the patient. The present application also discloses important advances in apheresis for therapeutic treatments, including treatments for sepsis and acute kidney injury using whole blood apheresis and immunotherapy, where targets impeding recovery are removed by apheresis and the previously removed engineered fragments are reintroduced. The use of selective removal through apheresis opens up the possibility of solving diseases and conditions previously thought to be untreatable.

Description

[Technical field]

[0001] Priority Data and Incorporation by Reference This application is a utility U.S. patent application claiming priority to U.S. Provisional Application No. 63 / 256,567, filed October 16, 2021. Although no other priority claims are made, this case relates to a family of cases relating to the treatment of mammals, including humans, relying in part or entirely on the technology of apheresis. Related cases include those relating to apheresis relying on selective removal of targets such as galectin-3, as disclosed in U.S. Pat. No. 8,764,695. This application is also related to U.S. Pat. No. 10,953,148, which relates to an apparatus for performing such apheresis. The subject matter of this application also relates to patents such as U.S. Pat. No. 11,389,476, which relate to methods of treating mammals for sepsis using apheresis.

[0002] 2. Background of the Invention FIELD OF THEINVENTION As alluded to above, the present application relates to the treatment of mammalian patients using apheresis, which may include the use of selective removal of targeted compounds, such as galectin-3. Selective removal refers to the use of targeted binding agents, such as antibodies, chemical binding agents, such as modified citrus pectin, or natural ligands, such as tumor necrosis factor alpha, and inhibitors, such as PDL-1 / 2 inhibitors, which may be presented in a column, filter, or other passageway of the apheresis device, whereby blood flowing through the device is exposed to the binding agent that selectively removes targets from the blood, which may be proteins, such as galectin-3, or proteins that interfere with the body's mechanisms for dealing with immune threats, such as tumor necrosis factor alpha or PDL-1 / 2. The present application details a strategy for apheresis using whole blood, rather than requiring separation of plasma from other blood components, such as blood cells and platelets. This simplifies the process considerably, making it easier to tolerate, less expensive, and more broadly applicable to individual patients and procedures. This application also describes opportunities for immunotherapy using apheresis (whole blood or plasma only), opened up in part by these new advances.

[0003] Summary of the Invention The present invention discloses and presents the actual treatment of blood of a mammalian patient using apheresis, but without separation or pretreatment into fractions such as platelets, plasma, whole cells, etc. This dramatically simplifies the apheresis process, making it easier and more manageable for the patient. The process is performed without separation of blood fragments, or by otherwise conditioning the mammal prior to apheresis. This makes the performance of apheresis cheaper and less time consuming. It also reduces the stress and difficulties encountered in the prior art implementations, without loss of efficacy, especially in the context of apheresis performed with selective removal of targets such as galectin-3 or other blood components. In this application, apheresis performed on whole blood is simply referred to as whole blood apheresis. This is distinguished from the prior art processes in which blood is diverted from the body and then separated into components, plasma, white blood cell, platelet fractions, etc. In "whole blood apheresis," as that term is used herein, blood is diverted from the body but introduced directly into an apheresis machine, where elements may be removed from the blood and other elements may be introduced into the patient's blood, which is then returned to the body.

[0004] Detailed Description of the Invention Whole blood apheresis was demonstrated to be effective in the course of a study evaluating the efficacy of rats with sepsis induced by the well-established model cecal ligation and puncture-induced sepsis (CLP). Rodent cecal ligation and puncture is considered the gold standard in sepsis research and the most widely used model for experimental sepsis. Developed more than 30 years ago, cecal ligation and puncture is considered a realistic model for the induction of polymicrobial sepsis to study the underlying mechanisms. Cecal ligation and puncture is characterized by midline laparotomy (accessed through a mid-abdominal incision) followed by needle puncture of the cecum, ligating the sphincter below the ileocecal valve, at the ileal junction (last part of the small intestine), and the colon (first part of the large intestine). As the cecum is a source of endogenous bacterial contamination, perforation of the cecum leads to bacterial peritonitis with subsequent translocation of mixed enteric bacteria into the blood system. During the onset of sepsis, bacteremia then triggers the activation of a systemic inflammatory response, followed by septic shock, multiple organ dysfunction, and death. When cecal ligation and puncture models are used in rodents, they exhibit a disease pattern with typical symptoms of sepsis or septic shock, such as hypothermia, tachycardia, and tachypnea.

[0005] Sepsis is the leading cause of death in intensive care units (ICUs) worldwide and the most common cause of acute kidney injury (AKI) in modern times. Across resource-rich and resource-poor settings, sepsis and sepsis-related acute kidney injury (S-AKI) are associated with significant morbidity and mortality as well as high healthcare costs. The annual incidence of sepsis in the United States (USA) exceeds 1.7 million cases and accounts for one in three in-hospital deaths. Furthermore, sepsis-related acute kidney injury disproportionately contributes to sepsis mortality and severe morbidity, accounting for more than half of sepsis-related deaths. In survivors of sepsis-related acute kidney injury, renal dysfunction increases the risk of chronic kidney disease (CKD) and remains a significant factor affecting long-term disability, quality of life, and survival.

[0006] Sepsis is a potentially fatal complex immune disorder resulting from dysregulation of multiple host defense pathways in response to infection. Sepsis is characterized by a massive release of cytokines, among other inflammatory mediators, that lead to fatal organ injury. In the United States, the incidence of sepsis and sepsis-associated acute kidney injury remains high, with a dramatic increase in the incidence of acute kidney injury among patients admitted to tertiary care hospitals from 7.2% in 2002 to 20% in 2012. 5 Current treatment of sepsis-associated acute kidney injury is limited to antibiotic therapy and organ support, which includes the provision of hemodialysis or continuous renal replacement therapy. There are no approved therapies that prevent, halt the progression, or promote recovery after sepsis-associated acute kidney injury. Novel therapeutic interventions in the management of sepsis and sepsis-associated acute kidney injury remain a significant unmet need.

[0007] Galectin-3 (Gal-3) is a member of a family of soluble 32-35 kilodalton (kDa) lectin proteins. Gal-3 is expressed in most human tissues, including an array of immune cells (such as macrophages, dendritic cells, eosinophils, mast cells, natural killer cells, activated T cells, and activated B cells), epithelial cells, endothelial cells, and sensory neurons. The scientific literature has identified Gal-3 as a driver of proinflammatory and profibrotic signaling in a wide variety of acute and chronic diseases, including sepsis, acute kidney injury, chronic kidney injury, heart failure, nonalcoholic steatohepatitis (NASH), idiopathic pulmonary fibrosis (IPF), and autoimmune diseases, as well as an oncogenic protein in tumor development. In response to infection and toxic insults, Gal-3 functions as an "alarmin" that instigates the immune response. Gal-3 is upregulated, brought to the cell surface, and secreted into the circulation. Gal-3 activates membrane toll-like receptors and ignites intracellular inflammasome protein complexes, resulting in cytokine release, hyperinflammation, and immune dysregulation. Notably, inflammasome activity has been shown to be responsible for pulmonary inflammation and acute respiratory distress syndrome, which causes both higher mortality and reduced bacterial clearance in the setting of coronavirus disease 2019 (COVID-19), influenza, and bacterial superinfection. Furthermore, by forming a cell surface lattice structure that is a ligand-Gal-3 complex and binding bioactive glycoproteins and glycolipids, Gal-3 fuels excessive inflammation and fibrosis, which contributes to renal dysfunction and failure.

[0008] Multiple studies from our group and others indicate that Gal-3 is not just a biomarker but plays a concerted causal role in the pathogenesis of sepsis and sepsis-associated acute kidney injury. In a mouse model of sepsis secondary to pulmonary infection, Gal-3 was upregulated and secreted into the extracellular space and circulation in septic mice. Elevated serum Gal-3 concentrations were associated with an exaggerated inflammatory response, cell death, and increased vascular damage. Gal-3 knockout (KO) mice demonstrated reduced lung pathology and significantly improved survival compared to wild-type mice (p=0.0003). Furthermore, Gal-3 knockout mice showed reduced inflammation and tissue injury, as well as significantly lower levels of inflammatory markers, inflammatory mediators, and vascular damage markers, such as C-reactive protein (CRP), interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF), thrombopoietin, and fibrinogen.

[0009] We have demonstrated the role of Gal-3 in sepsis and sepsis-associated acute kidney injury through both oral Gal-3 inhibition and removal of Gal-3 by apheresis in a rat model. In a recent study published in Critical Care, we investigated 7-day mortality, serum Gal-3, IL-6, and creatinine concentrations in a cecal ligation and puncture (CLP) rat model of sepsis and sepsis-associated acute kidney injury. 27 Both serum Gal-3 and IL-6 were significantly elevated after cecal ligation and puncture. Rats pretreated with oral Gal-3 inhibitors at 400 mg / kg / day and 1200 mg / kg / day before the cecal ligation and puncture procedure had significantly decreased serum concentrations of both Gal-3 and IL-6 compared to controls. Notably, circulating Gal-3 levels were consistently elevated and spiked earlier than IL-6, indicating its role as an upstream mediator in the inflammatory cascade in sepsis and sepsis-associated acute kidney injury. Seven-day mortality was significantly lower in the 400 mg (28%, p=0.03) and 1200 mg (22%, p=0.001) Gal-3 inhibitor groups compared to controls (61%). Furthermore, the incidence of acute kidney injury was significantly reduced from 89% in the control group to 44% (p=0.007) in both Gal-3 inhibitor groups based on the RIFLE (Risk of Renal Impairment, Damage to the Kidneys, Failure or Deterioration of Renal Function, and End-Stage Renal Disease) criteria. The oral Gal-3 inhibitor used in these studies was Pectasol® Modified Citrus Pectin (P-MCP), a low molecular weight pectin that directly inhibits Gal-3 by binding to its glycan recognition domain. The lead investigator developed Pectasol® as a dietary supplement, and as a pectin it is classified as generally regarded as safe (GRAS) by the US Food and Drug Administration. The efficacy of P-MCP has been confirmed in multiple conditions and animal models. In a companion study evaluating septic patients, serum Gal-3 on admission to the intensive care unit was an independent predictor of intensive care unit mortality (p=0.04) and acute kidney injury (p=0.01).We have recently been able to perform rat Gal-3 depletion apheresis in a cecal ligation and puncture model. We demonstrated a significant difference in survival between the Gal-3 apheresis group (survival rate: 9 / 10 rats) and the sham apheresis group (survival rate: 1 / 9 rats) (p<0.01). We discuss this study in more detail in the landmark chapter below.

[0010] In a study of ischemia / reperfusion (I / R) injury using a renal pedicle occlusion mouse model, Gal-3 knockout mice showed significantly reduced acute renal tubular necrosis (p<0.0001) and enhanced renal tubular regeneration (p<0.005) compared to controls. Furthermore, Gal-3 knockout mice showed significantly lower levels of IL-6 (p<0.05) and IL-1β (p<0.05), as well as reduced reactive oxygen species (p=0.003). In most of our recent rat model studies of Gal-3 in ischemia / reperfusion injury, Gal-3 and IL-6 were significantly elevated from baseline after renal pedicle occlusion, with Gal-3 levels elevated before IL-6. Pretreatment with 36Gal-3 inhibitors resulted in significantly reduced serum Gal-3 and IL-6, renal tubular injury, and apoptosis, and improved renal function (p<0.05). In a companion study of 52 patients admitted to the intensive care unit after coronary artery bypass grafting (CABG) without pre-existing renal disease, serum Gal-3 concentration at intensive care unit admission was an independent predictor of acute kidney injury and performed better than neutrophil gelatinase-associated lipocalin (NGAL), cystatin C (CycC), and serum creatinine (Cr) as an early biomarker of acute kidney injury (area under the receiver operating characteristic curve [AUC-ROC]: Gal-3 0.890; NGAL 0.763; Cr 0.773). It is important to note that in human studies, the elevation of serum Gal-3 persists for a longer period of time. For example, in an observational study of 645 intensive care unit patients with incident acute kidney injury, serum Gal-3 levels remained elevated at the time of hospital discharge, and Gal-3 levels correlated with the severity of acute kidney injury.

[0011] Inhibition of Gal-3 has been demonstrated to reduce inflammation and prevent renal fibrosis in multiple mouse models of acute kidney injury. In a mouse study utilizing a folic acid-induced kidney injury model, mice were treated with an oral Gal-3 inhibitor one week prior to folic acid injection. The Gal-3 inhibitor group demonstrated a significant reduction in acute massive kidney enlargement. Pretreated mice demonstrated a 30% reduction in Gal-3 protein expression two weeks after folic acid injection. Pretreatment with Gal-3 inhibitor significantly reduced renal fibrosis (p<0.05) as well as the levels of fibrosis markers (collagen I, fibronectin, and transforming growth factor beta [p<0.05]), proinflammatory cytokines (IL-1b [p<0.05] and tumor necrosis factor alpha [p<0.05]), and apoptosis (p<0.01). 30 In other studies, Gal-3 inhibitors successfully reduced inflammation and fibrosis in multiorgan injury and disease models. Notably, in patients with renal dysfunction, elevated serum Gal-3 is associated with rapid deterioration of renal function, incident chronic kidney disease, and all-cause mortality.

[0012] Gal-3 depletion in a sepsis model is unprecedented, differentiating our approach from endotoxin removal and other ex vivo strategies. Potential future applications include acute kidney injury of other etiologies, chronic kidney disease, nonalcoholic steatohepatitis, as well as enhanced immunotherapy in cancer, heart failure, myocardial infarction, and idiopathic pulmonary fibrosis.

[0013] Finally, several groups have published studies showing that elevated serum levels of Gal-3 predict progression to severe COVID-19 in patients infected with SARS-CoV-261,62, suggesting that Gal-3 is an attractive upstream target to regulate inflammatory responses and prevent cytokine storm syndrome in these patients. Therefore, our focus is on sepsis / acute kidney injury, and the potential of Gal-3 depletion therapy to treat acute COVID-19 provides further urgency for our application.

[0014] Taken together, multiple studies have demonstrated a concerted role for Gal-3 in the pathogenesis of sepsis and acute kidney injury using multiple methods including pharmacological oral inhibitors and knockout mice, as well as observational data in humans. These studies are consistent with a critical role for Gal-3 in accentuating inflammatory and fibrotic responses to acute injury. With evolving evidence consistent with a causal role for Gal-3 in sepsis and sepsis-related acute kidney injury, and the urgent need for therapeutic intervention, we have proposed Gal-3-specific apheresis as a novel treatment for sepsis and sepsis-related acute kidney injury. We reason that rapid and efficient depletion of excess plasma Gal-3 will suppress and potentially reverse the immune dysregulation underlying sepsis, reducing both morbidity and mortality in sepsis and sepsis-related acute kidney injury. The proposed project addresses the urgent need for practical, fast-acting therapeutic interventions that can be implemented in patients with sepsis and sepsis-related acute kidney injury.

[0015] We herein disclose a novel treatment for sepsis and sepsis-associated acute kidney injury through the depletion of serum Gal-3 using our proprietary Gal-3 selective apheresis column, XGal3®. Our proposal includes several innovative components. This unique medical device incorporates the first selective Gal-3 adsorbent capture molecule into an apheresis column. Gal-3 depletion apheresis is a novel product and procedure invented by Principal Investigator Dr. Eliaz, who developed the first commercially available Gal-3 inhibitor and has been engaged in Gal-3 research and clinical applications for 25 years. Over the past six years, in collaboration with leading experts around the world, our team has developed a proprietary monoclonal Gal-3 capture antibody that selectively binds Gal-3. The Gal-3 apheresis column is compatible with clinical apheresis systems currently used in hospitals and clinics, simplifying the regulatory and commercialization pathways. XGal3® filters are compatible with pharmaceutical treatments as well as additional / other extracorporeal therapies.

[0016] Gal-3 specific therapeutic apheresis has the potential to reduce morbidity and mortality associated with sepsis and sepsis-associated acute kidney injury, a condition for which there are no effective treatments. Furthermore, our novel approach also has the potential to reduce the deterioration of renal function and prevent or ameliorate chronic kidney disease in sepsis survivors.

[0017] Therapeutic apheresis offers an effective and safe treatment option compared to drug treatment. Pharmacological interventions are limited by pharmacokinetics, drug-drug interactions, toxicity, and other adverse effects. These limitations become increasingly more complex in critically ill patients. The Gal-3 selective apheresis column XGal3® offers the possibility to rapidly and safely remove Gal-3 from the circulation without toxicity, side effects, and dose limitations. Furthermore, Gal-3 specific apheresis can be performed repeatedly and as frequently as necessary. Notably, Gal-3 is rapidly regenerated at the cellular / tissue level, and depletion, inhibition, and knockout of Gal-3 show no adverse effects in animal models or humans.

[0018] Gal-3 functions by generating a pentameric complex that crosslinks with target ligands. All Gal-3 inhibitors developed function as competitive inhibitors at the carbohydrate recognition domain (CRD) and are therefore limited to blocking Gal-3. In contrast, the XGal3® antibody binds to the Gal-3 pentamer at the N-terminus, allowing the antibody to remove pathogenic ligand-bound Gal-3 monomers and pentamers from the circulation. Oral Gal-3 inhibitors are in development, but none have been tested for use in sepsis and sepsis-associated acute kidney injury. GS-100, a form of modified citrus pectin developed by La Jolla Pharmaceuticals, was initially targeted for the treatment of chronic kidney injury but was discontinued for economic reasons. Unlike the rapid and efficient clearance provided by XGal®, the efficacy of pharmacological inhibitors is contingent on the strength, specificity, metabolism, potency, and side effect profile of the Gal-3-ligand interaction. Furthermore, Gal-3 inhibitors may compete with endogenously bound ligands for the glycan recognition domain, leading to off-target effects through binding to other galectins. In contrast, the design of the XGal3® column allows for selective and rapid removal of Gal-3 from plasma without competition for ligand binding, drug-related complications, or off-target effects.

[0019] Extracorporeal treatments for sepsis have included therapeutic plasma exchange (TPE) and filtration columns. A 2014 meta-analysis of four randomized controlled trials (RCTs) showed no association with overall mortality with TPE. European approval of Cytosorb® (CytoSorbents Europe GmbH, Berlin, Germany) apheresis columns for removal of IL-6, IL-10, and tumor necrosis factor has progressed but with limited success. Polymyxin B cartridges in extracorporeal hemoperfusion devices (PMX-DHP, Toray Medical Co., Tokyo, Japan) are the treatment of choice in Japan and Western Europe for removal of endotoxins. During the COVID-19 pandemic, both Cytosorb® columns and polymyxin B devices received U.S. Food and Drug Administration emergency use authorization in the United States for use in critically ill COVID-19 patients. However, neither treatment has demonstrated a significant effect on survival rates to date. Other extracorporeal strategies include large volume hemofiltration, hemoadsorption, combined plasma filtration and adsorption, high cut-off membranes, and hemoperfusion. Continuous hemodialysis, which uses a polymethylmethacrylate (PMMA) membrane hemofilter (PMMA-CHDF, Toray Medical Co., Tokyo, Japan) to remove multiple pro- and anti-inflammatory cytokines, has shown conflicting and limited results in the treatment of sepsis in clinical studies. In a meta-analysis of randomized controlled trials using hemoperfusion with polymyxin B, the authors found no effect on 28-day mortality. These developments demonstrate the urgent need for effective therapies for the treatment of sepsis and the growing interest in apheresis as a therapeutic approach for sepsis. Many other investigators have tried and failed to develop effective apheresis-based therapies for sepsis, but they all relied on nonspecific adsorption and elimination of a wide variety of pro- and anti-inflammatory mediators. Our approach is fundamentally different in that we target an upstream mediator of the inflammatory response (Gal-3), a novel target for apheresis that we believe will be more effective.Our approach and the specific immunoprecipitation method allow us to combine Gal-3 depletion with other apheresis and filtration columns and devices if necessary, for example, Gal-3 depletion can be combined with renal replacement therapy (RRT) in patients with sepsis-associated acute kidney injury in the intensive care unit.

[0020] The inventors have completed important milestones: demonstrating depletion of Gal-3 from serum using an antibody (Ab); publishing a proof-of-concept (POC) study in a porcine skin inflammatory injury model; developing a successfully immobilized proprietary anti-Gal-3 antibody; and developing an apheresis column that efficiently removes Gal-3. We have established the time course of serum Gal-3 and serum IL-6 concentrations in a septic rat circulation model; performed therapeutic apheresis in healthy rats; demonstrated that inhibition of Gal-3 efficiently reduced serum Gal-3 and systemic inflammation, protected against sepsis-associated acute kidney injury, and enhanced survival in a rat model of sepsis; successfully completed a proof-of-concept study using a cecal ligation and puncture rat model of sepsis and sepsis-associated acute kidney injury in which removal of Gal-3 from the circulation dramatically reduced mortality; and developed a prototype Gal-3-selective apheresis column for human clinical use.

[0021] Examples of whole blood apheresis The present inventors screened commercially available anti-rat Gal-3 antibodies, but none of them performed sufficiently well. The present inventors developed a new high-affinity anti-rat Gal-3 antibody using rabbit and rat Gal-3 antigens, and estimated the affinity by evaluating the top 8 positive clones from a concentration-adjusted ELISA plate immobilized with recombinant rat Gal-3. The top clones were then evaluated using surface plasmon resonance (SPS). The equilibrium dissociation constant (KD) of the highest affinity clone was 2.889 × 10 -10 And this is more than enough.

[0022] After we developed a novel anti-rat Gal-3 antibody, we successfully coupled it to Sepharose beads and made a 0.4 ml mini-column and pseudo-mini-column of activated resin.

[0023] We then attempted to perform a critical efficacy study to evaluate the effect of Gal-3 apheresis on the survival rate of rats subjected to cecal ligation and puncture. Unfortunately, the prolonged apheresis procedure with reduced flow rate and plasma separation performed only 1 hour after the cecal ligation and puncture procedure was too harsh for the rats, and all animals in both the sham and active groups did not survive the procedure.

[0024] Therefore, we performed whole blood apheresis / filtration using the same mini-column. As a result, we were finally able to complete the originally proposed Gal-3 apheresis depletion study in 19 rats (10 using the active Gal-3 depletion column and 9 using the sham blank column), with apheresis performed for 90 min 1 h after cecal ligation and puncture.

[0025] The minicolumn used was packed with 0.4 ml of activated Sepharose containing 2 mg / ml of our anti-rat gal-3 antibody. The flow rate was 0.5-0.8 ml / min.

[0026] Nine out of ten treated rats survived to the pre-specified 7-day endpoint, compared to only one out of nine rats in the sham-treated control group. (All surviving animals were euthanized on day 7 as per protocol). This novel result is a dramatic and significant (p<0.001) demonstration that Gal-3 apheresis is effective in attenuating sepsis. Ex vivo studies were performed to confirm the ability of anti-Gal-3 (rat) antibodies to deplete Gal-3. Additional rats were subjected to renal ischemia-reperfusion injury (I / R), plasma was collected 2 hours after reperfusion, and ex vivo depletion was performed on the active column. Ex vivo studies confirmed that the active column depleted Gal-3 levels (79% vs. 2% for the sham column). It is important to note that our anti-Gal-3 (rat) antibody is less effective than our anti-Gal-3 (human) antibody (>90%). Therefore, we expect the treatment to translate well to humans, who will better tolerate the apheresis treatment and can receive replacement fluids if necessary.

[0027] Opportunities for Immunotherapy Among the many applications that lend themselves to apheresis and could be improved in both efficacy and simplicity through whole blood apheresis are immunotherapies. A wide range of existing treatments and technologies are discussed, including treatment with tumor infiltrating lymphocytes (TILs), CAR-T cells, stem cell induction and stem cell infusions, such as PD-1 inhibitors, which typically target various forms of cancer. All of these therapies could be improved using apheresis. Currently, much focus is placed on the use of PD-1 and PDL-1 inhibitors to make cancer treatments more effective. Apheresis could dramatically enhance these treatments using the technology described herein, which could include whole blood apheresis or apheresis with plasma separation.

[0028] Thus, rather than simply relying on the administration of drugs that inhibit PD-1 and PDL-1 (inhibitors), now the PD-1 and PDL-1 material can be removed from the blood by passing the blood through an apheresis device or column and passing the blood through an antibody (or other ligand) in the apheresis column specific for PD-1, and then the blood can be returned to the patient, thereby reducing the interference presented by PD-1. Treatment can be augmented by the administration of inhibitors introduced into the blood before returning it to the patient, or preferably as soon as possible after the end of the apheresis treatment, ideally. Treatment with tumor-infiltrating lymphocytes, CAR-T cell immunotherapy, and the derivation and recovery of stem cells all require the removal of target cells or target material from the patient. The target is often genetically modified and then reintroduced into the body. This procedure can be simplified and enhanced by the use of apheresis, both for the recovery and for the administration of materials such as stem cells and T cells for CAR-T immunotherapy, tumor-infiltrating lymphocytes. In these methods, the harvested materials are collected and genetically modified. They must then be returned to the patient. All these methods can be performed using apheresis, either whole blood apheresis or plasma separation-based apheresis, and the procedure becomes faster, easier, and more effective in that the selective removal can be combined with the administration of additional materials to enhance efficacy. For example, soluble PD-L1, which has PD-1 binding ability, is present in the plasma of patients with cancer, such as non-small cell lung cancer. PD-L1 is one of the important immune checkpoint molecules that can be targeted by cancer immunotherapy. PD-L1 has a soluble form (sPD-L1) and a membrane-bound form (mPD-L1). If we remove soluble PD-L1 (sPD-L1) by gradient equilibration, we can expect that not only will the expression of mPD-L1 decrease, but mPD-L1 will be released into the blood, and thus the presence of sPD-L1 will increase. In this way, apheresis of whole blood or plasma can deplete not only soluble but also membrane PD-L1, which may allow for better responses to different PD-L1 inhibitors and may also allow for reduced doses with less toxicity.Simultaneous or sequential removal of galectin-3 and related compounds such as inflammatory cytokines, e.g., IL1B, IL-6, IL-4, IL-8, tumor necrosis factor alpha, nuclear factor kappa beta, and others, can further enhance the efficacy of immunotherapy while addressing its inflammation-based toxicity. A similar approach can be utilized pre- or post-dialysis for patients with end-stage renal disease, chronic kidney disease, various autoimmune conditions, and patients with sepsis, acute kidney injury, sepsis-associated acute kidney injury, and other life-threatening conditions. It can be used for patients with retinal nerve fiber layer defects, nonalcoholic steatohepatitis, peripheral arterial disease, coronary artery disease, and toxic burdens of various etiologies.

[0029] Given the methods and procedures presented herein, one skilled in the art can modify the parameters of apheresis to meet the needs of the patient and the requirements of the equipment. Process metrics such as blood flow, column size, and residence time can be modified based on the condition(s) being treated and the number / amount of targets to be removed or isolated. A typical size column for a whole blood column would be 40-500 ml, perhaps about 100-200 ml. Membrane technology or various highly resistant resins can be used as the matrix, which is activated with ligands that target the compounds to be removed. Plasma separation and cell recovery may also be used before, during, or after compound removal. It is preferred to remove specific cells before removal of targeted compounds. As is the case with the size and number of channels or columns, the blood flow can be run by one skilled in the art based on availability and need. If the device / platform used is a dialysis machine, it will require a central line capable of drawing volumes greater than 100-300 ml / min, with sufficiently wide tubing / lumens (4 French), a double lumen central line catheter, and special ports (BARDA and Angiodynamic are two well-known brands). Residence times can vary from 30-300 seconds. Flow rates require sufficiently high blood flow rates to prevent blood cell clumping if whole blood apheresis is being performed. Membrane technology is preferred for whole blood, although highly resistant resins may also work. Diameters are typically 3-10 cm based on volume, matrix, and desired blood / plasma flow rates. This is well known to those skilled in the art.

[0030] This application discloses the use of whole blood apheresis as an effective means of treating mammalian patients for sepsis and related conditions, as well as various immunotherapeutic applications. This application also discloses the use of whole blood apheresis for the treatment of mammalian patients and conditions. The ability to treat mammals, including humans, through whole blood apheresis for a wide variety of diseases and treatments, including but not limited to sepsis and acute kidney injury, would pave the way for treatment for a wide variety of conditions, with lower costs and fewer barriers, and through a process that is applicable to a wide variety of individuals and situations. Of the many treatments that have become more effective, immunotherapy has been amenable to this approach.

Claims

1. An agent that binds to galectin-3 for use in a method of treating sepsis in a mammalian patient, wherein The method includes performing whole blood apheresis in which blood is directed from the mammalian patient to an apheresis machine, the agent selectively recovering galectin-3 from the patient's blood; and The blood is returned to the patient after selective collection without blood separation. Drugs.

2. An agent that selectively removes galectin-3 for use in enhancing the treatment of a mammal by immunotherapy, wherein the treatment comprises performing whole blood apheresis on the mammal to remove a portion of the mammal's blood, selectively removing from the portion of the blood an agent selected from the group consisting of PD-1, PDL-1, tumor infiltrating lymphocytes, T cells for chimeric antigen receptor modification, and stem cells, and returning the blood to the patient after the selective removal apheresis. Drugs.

3. The method of claim 2 , wherein the immunotherapy further comprises the administration of an anti-cancer agent effective in treating one or more types of cancer.

4. 4. The method of claim 3, wherein the administration of the anti-cancer agent is accomplished simultaneously with or close to the apheresis.