WO2021214588A1 - Anti-tnf alpha agent for treating coronavirus infections - Google Patents

Abstract

An anti-TNFa agent that blocks binding of TNFa to TNFa receptor, for example, an anti-TNFa antibody such as infliximab is useful in the treatment or prevention of infection of a human with SARS-CoV-1 and/or SARS-CoV-2.

Description

ANTI-TNF ALPHA AGENT FOR TREATING CORONA VIRUS INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Number 63/013,004, filed April 21, 2020, the entire disclosure of which is hereby incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submitted electronically via EFS- Web as an ASCII formatted sequence listing with a file name “JBI6305WOPCTlSeqFisting4-8- 21.txt”, creation date of April 8, 2021, and having a size of 3KB. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

Introduction

The invention relates to methods of treating and/or preventing SARS-CoV-1 and/or SARS-CoV-2 infection with an agent that binds TNFa. The invention also relates to the use of an anti-TNFa agent in the manufacture of a medicament for the treatment, prevention and/or inhibition of SARS-CoV-1 and/or SARS-CoV-2 infection. The invention further relates to a dosing regimens for the administration of an anti-TNFa agent treat and/or prevent infection SARS-CoV-1 and/or SARS-CoV-2 infection, the method comprising administering a therapeutically effective amount of an anti-TNFa agent to a patient pursuant to the clinical protocol and parameters described herein.

Background

Coronaviruses infect a variety of host species, including humans and several other animal species. In their natural hosts, these viruses predominantly cause respiratory and intestinal tract infections producing a wide range of clinical manifestations. Coronaviruses infecting the respiratory tract have long been recognized as significant pathogens in animals and as the cause of mild and severe respiratory illness in humans. Highly pathogenic human coronaviruses such as SARS-CoV and MERS-CoV similarly cause a wide spectrum of clinical manifestations in humans. Typically, the majority of patients develop a short period of moderate clinical illness; however, a small but a substantial number of patients develop severe pneumonia often associated with rapid virus replication, massive inflammatory cell infiltration and elevated proinflammatory cytokine/chemokine responses resulting in ALI, and acute respiratory distress syndrome (ARDS). It has been well documented for many years that infection by several members of the Coronaviridae family both in vitro and in vivo in humans and animals is associated with increases in the production of TNFa. This induction of TNFa expression can be caused by both productive viral infection, non-productive infection, noninfectious virus-like particles, and even individual coronavirus proteins such as the spike or nucleocapsid proteins.

Coronaviruses cause disease in a number of mammals and birds. Coronaviruses are enveloped viruses having positive-sense single-stranded RNA genomes and are classified as members of the Orthocoronavirinae subfamily, which includes the Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus genera. In humans, they have been responsible for important recent outbreaks of infectious diseases, including the Severe Acute Respiratory Syndrome (SARS) outbreak of 2002-2003, the Middle East respiratory syndrome (MERS) outbreaks occurring since 2012, and the coronavirus disease 2019 (COVTD-19) pandemic of 2019-2020. SARS, MERS and COVTD-19 are all caused by novel coronaviruses (SARS-CoV-1, MERS-CoV and SARS-CoV-2, respectively). The SARS pandemic developed rapidly with the consequence that treatments lacked randomised placebo-controlled clinical trials. Despite this, a combination of ribavirin and corticosteroids was used in Hong Kong, Canada and elsewhere, as well as a pulse methylprednisolone therapy dosed at 250 to 500 mg/day for 3 to 6 days in critically ill patients infected with the virus. There are currently no approved vaccines or antiviral drugs for patients infected with MERS-CoV or SARS-CoV-2.

SARS-CoV-2, the causative agent of COVTD-19, is an enveloped, positive-sense, single- stranded RNA betacoronavirus. It was first identified following reports of a cluster of acute respiratory illness cases in Wuhan, Hubei Province, China in December 2019. Epidemiological investigations indicated that the majority of early cases were linked to a seafood market, with patients infected through zoonotic or environmental exposure, followed by the subsequent spread of infection by human-to-human transmission among close contacts. Genomic sequencing was performed on bronchoalveolar lavage fluid samples collected from patients with viral pneumonia admitted to hospitals in Wuhan, which identified a novel RNA virus from the family Coronaviridae. Phylogenetic analysis of the complete viral genome revealed that the virus, SARS- CoV-2, is part of the subgenus Sarbecovirus of the genus Betacoronavirus, and is most closely related (approximately 88% identity) to a group of SARS-like coronaviruses previously sampled from bats in China. Since the novel SARS-CoV-2 virus was observed in humans in late 2019, hundreds of thousands have been infected and thousands have died as a result of the associated disease, termed COVID-19. Symptoms of infection may appear from 2 to 14 days following exposure, with the spectrum of illnesses ranging from mild symptoms to severe illness or death. Severe clinical presentations have been reported for as many as 20-25% of laboratory-confirmed cases. In a study of 99 patients in a single center in Wuhan with SARS-CoV-2 infection confirmed by real-time reverse-transcriptase polymerase chain reaction (RT-PCR), the most commonly reported clinical manifestations were fever (83%), cough (82%), shortness of breath (31%), and muscle ache (11%). In chest x-rays and CT scans, 75% of patients showed bilateral pneumonia and 14% of patients showed multiple mottling and ground-glass opacities. In a further study of 138 patients with novel coronavirus-induced pneumonia in a single center in Wuhan, common symptoms included fever (98.6%), fatigue (69.6%), and dry cough (59.4%). Lymphopenia occurred in 70.3% of patients, and chest CT scans showed bilateral patchy shadows or ground-glass opacities in the lungs of all patients. Thirty-six patients (26%) were transferred to the ICU because of complications, including ARDS, arrhythmia, and shock. Broadly, similar findings were noted in other case studies, e.g., in the Seattle region in the US. At present, it appears that individuals aged 65 years or older, especially those with comorbid diseases, are subject to the highest incidence of morbidity and mortality. In contrast, a study of 2,143 children aged <18 years in China with laboratory confirmed (34.1% of cases) or suspected (65.9% of cases) COVID-19 indicated that the clinical manifestations of the disease may be less severe in children than adults, with approximately 94% of cases being asymptomatic, mild, or moderate. However, young children, particularly infants, were susceptible to severe disease, with the highest proportion of severe and critical cases by age group reported for children aged <1 year (10.6% of cases) and 1-5 years (7.3% of cases). Also, new evidence has emerged from China indicating that a large number of infections do not result in symptoms.

Learnings from SARS-CoV-2, initial COVID-19 data and preclinical mouse model data indicate that many cytokines including TNFa, IL-1, and TNFa, may be key drivers of the acute lung injury (ALI) and ARDS observed in COVID-19. Severe COVID-19 patients from an initial study in China had elevated levels of TNFa. SARS-CoV-2 has been directly implicated in stimulating TNFa generation. The virus' spike protein has been shown to interact with angiotensin-converting enzyme 2 (ACE2) with consequent activation of TNFa converting enzyme (TACE), in a process that facilitates both viral entry into host cells and TNFa production. Excess TNFa has been associated with multiple inflammatory diseases and may be an initiating or contributing factor to disease pathology and progression. TNFa has been reported to be an amplifier of inflammation, and suppressing it has the potential to suppress multiple downstream cytokines such as IL-1 and TNFa, as well as decrease capillary leak, and leukocyte trafficking as has been shown in rheumatoid arthritis (RA) patients.

At the time of drafting, the global death toll is over 145,000. SARS-CoV-2 and coronaviruses more generally lack effective treatment, leading to a large unmet medical need. Current management of COVTD-19 is supportive, and respiratory failure from ARDS is the leading cause of mortality. While the understanding of the epidemiology and clinical spectrum of COVTD-19 is still evolving during the ongoing pandemic, it remains highly unpredictable and the current knowledge of the disease burden highlights the urgent medical need to develop a treatment.

TNFa as a Putative Target

TNFa is considered a key inflammatory mediator that exhibits a wide variety of functional activities. Excess TNFa has been associated with multiple inflammatory diseases and may be an initiating or contributing factor to disease pathology and progression. These findings make TNFa a logical target in the treatment of diseases involving TNFa-mediated inflammation, however the unpredictability in the art requires thoughtful clinical assessments for whether anti- TNFa drugs will be effective to mitigate symptoms associated with SARS-CoV-2. The hypothesis is that binding of infliximab or a molecule with the same binding regions as Infliximab, to TNFa may inhibit or prevent the interactions of this cytokine with its cellular receptors and may prevent the deleterious effects caused by TNFa. A robust clinical assessment is required to establish the facts supporting that hypothesis.

TNFa exists as either a transmembrane form or a smaller piece of this is cleaved by a metalloproteinase and exists as a soluble form. The effects are mediated by binding to either of two receptors (TNFRl or TNFR2). TNFRl is expressed on most cell types and is activated by soluble TNF while TNFR2 is expressed primarily on immune cells and is generally activated by transmembrane TNF. Tumor necrosis factor exerts its pleiotropic effects via many mechanisms: by macrophage activation, differentiation and phagosome formation; activation of neutrophils and natural killer cells; and promotion of cell adhesion, apoptosis and cellular proliferation.

As an example of an anti-TNFa antibody, Infliximab neutralizes the biological activity of TNFa by binding with high affinity to the soluble and transmembrane forms of TNFa and inhibits the binding of TNFa to both the p55 and p75 TNF receptors. Infliximab does not neutralize TNF beta (lymphotoxin alpha), a related cytokine that uses the same receptors as TNFa. Biological activities attributed to TNFa include: induction of proinflammatory cytokines such as interleukin- 1 (IL-1) and TNFa, enhancement of leukocyte migration by increasing endothelial layer permeability and expression of adhesion molecules by endothelial cells and leukocytes, activation of neutrophil and eosinophil functional activity, induction of acute phase reactants and other liver proteins, as well as tissue degrading enzymes produced by synoviocytes and/or chondrocytes. In vitro studies have shown that infliximab can bind to the surface of cells expressing the transmembrane form of TNFa and this may lead to their subsequent lysis by complement or effector cells. Infliximab inhibits the functional activity of TNFa in a wide variety of in vitro bioassays using human fibroblasts, endothelial cells, neutrophils, B and T lymphocytes, and epithelial cells. However, no efficacy data are available on the use of infliximab to treat COVTD-19 induced ARDS or similar disorders.

While the expression of TNFa and other cytokines are a component of the normal immune response, excessive expression of cytokines such as TNFa and the ensuing immune inflammation can lead to disease pathology. This excessive systemic release of proinflammatory cytokines, termed a “cytokine storm”, can occur in a number of non-infectious conditions such as graft-versus-host disease (GVHD), CAR-T cell therapy, and ARDS as well as several infectious diseases such as sepsis, Ebola and other viral hemorrhagic fevers, avian influenza, and smallpox.

Elevated TNFa levels in the fluid of the lungs have been associated with a poor prognosis for individuals ARDS. In the case of SARS, increased levels of TNFa have been reported to be a prognostic indicator associated with more severe pulmonary disease in animal models. Learnings from SARS-CoV, initial COVTD-19 data and preclinical mouse model data indicate that TNFa might indeed be a key driver of the ALI and ARDS observed in COVTD-19. More severe COVTD-19 patients from an initial study in China had elevated levels of TNFa.

Therefore, a need exists in the art for an effective treatment and therapeutic dosing regimens for treating and/or preventing a coronavirus infection with an anti-TNF alpha agent. The invention herein provides such methods.

Brief Description of the Drawings

Figure 1 shows a schema of the infliximab COVTD-19 study design.

Summarv of the Invention

The present invention relates to anti-TNFa agents comprising molecules that bind to TNFa — the sequence of which is well known and in the public domain and is available at the NCBI gene number 7124. Specifically such agents would be capable of blocking or inhibiting an TNFa activity, such as binding of TNFa to TNFa Receptor (TNFaR), for use in the treatment or prevention of infection of a human with SARS-CoV-1 and/or SARS-CoV-2 (in particular, COVID-19). The present invention further relates to treatment strategies or protocols using anti- TNFa agents comprising molecules that bind to TNFa and block or inhibit an TNFa activity, such as binding of TNFa to TNFa Receptor (TNFaR), for use in the treatment or prevention of infection of a human with SARS-CoV-1 and/or SARS-CoV-2 (in particular, COVID-19). In an embodiment, the anti-TNFa agent is an anti-TNFa antibody or an isolated anti-TNFa antibody.

In another aspect, the invention relates to the use of an anti-TNFa agent in the manufacture of a medicament for the treatment or prevention of infection of a human with SARS-CoV-1 and/or SARS-CoV-2 (e.g., COVID-19). In such embodiments, references to the medicament being administered (e.g., “wherein the medicament is administered”) include the medicament being prepared for administration (and thus may be amended to “wherein the medicament is prepared for administration”) with the features in question. References to the anti-TNFa agent being administered (“anti-TNFa agent is administered”) are referring to the anti-TNFa agent within the medicament that is being administered to the human.

In another aspect, the invention relates to a method of using an anti-TNFa agent to treat or prevent infection of a human with SARS-CoV-1 and/or SARS-CoV-2, the method comprising administering a therapeutically effective amount of an anti-TNFa agent to the human.

In another aspect, the invention relates to an anti-TNFa agent in a package together with instructions for its use in the treatment or prevention of infection of a human with SARS-CoV-1 and/or SARS-CoV-2.

In another aspect, the invention relates to the inhibition of infection of a human cell with SARS-CoV-1 and/or SARS-CoV-2 by treating the cell with an anti-TNFa agent.

In another aspect, the invention relates to the inhibition of one or more viral activities in a human cell infected with SARS-CoV-1 and/or SARS-CoV-2 by treating the cell with an anti- TNFa agent.

In a preferred embodiment, the isolated antibody is infliximab or an antigen-binding fragment thereof. The sequence and structure of infliximab is described in U.S. 6,284,471 which is Continuation-in-part of application No. 08/010,406, filed on Ian. 29, 1993, now abandoned, and a continuation-in-part of application No. 08/013,413, filed on Feb. 2, 1993, now abandoned, which is a continuation-in-part of application No. 07/943,852, filed on Sep. 11, 1992, now abandoned, which is a continuation-in-part of application No. 07/853, 606, filed on Mar. 18, 1992, now abandoned, which is a continuation-in-part of application No. 07/670,827, filed on Mar. 18, 1991, now abandoned. Each of the foregoing applications and patents are expressly incorporated by reference. The light chain, variable region sequence of infliximab comprises the following amino acids from amino to carboxy terminus in single letter code:

(SEQ ID NO: 1 herein)

DILLTQSPAI LSVSPGERVS FSCRASQFVG SSIHWYQQRT NGSPRLLIKY ASESMSGIPS 60

RFSGSGSGTD FTLSINTVES EDIADYYCQQ SHSWPFTFGS GTNLEVK 107 and the heavy chain variable region sequence of infliximab comprises the following amino acids from amino to carboxy terminus in single letter code:

(SEQ ID NO: 2 herein)

EVKLEESGGG LVQPGGSMKL SCVASGFIFS NHWMNWVRQS PEKGLEWVAE IRSKSINSAT 60

HYAESVKGRF TISRDDSKSA VYLQMTDLRT EDTGVYYCSR NYYGSTYDYW GQGTTLTVS 119

These sequences are further provided in US 6,284,471 in Figure 16 and in SEQ ID NO: 3 (light chain) and SEQ ID NO: 5 (heavy chain) and are incorporated herein by reference.

In a preferred embodiment, the isolated antibody is administered intravenously (IV) at a dose of 1 to 10 mg per kg weight of the patient, preferably 3 to 8 mg per kg weight of the patient, more preferably 5 mg per kg weight of the patient. Preferably, the isolated antibody is infliximab and is administered intravenously (IV) at a dose of 5 mg/kg. Preferably, the isolated antibody is administered intravenously (IV) one time per day as a single IV dose, though additional dosing paradigms may be used and a subject is evaluated for improvement of symptoms pursuant to the clinical assessment parameters described herein.

Detailed Description of the Invention

Preferred embodiments of the invention are defined below. These preferred embodiments are applicable to all of the aspects of the invention defined herein.

The Coronavirus family contains the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus . All of these genera contain pathogenic viruses that can infect a wide variety of animals, including birds, cats, dogs, cows, bats, and humans. These viruses cause a range of diseases including enteric and respiratory diseases. The host range is primarily determined by the viral spike protein (S protein), which mediates entry of the virus into host cells. Coronaviruses that can infect humans are found both in the genus Alphacoronavirus and the genus Betacoronavirus. Known coronaviruses that cause respiratory disease in humans are members of the genus Betacoronavirus. These include SARS-CoV-1, SARS-CoV-2 and MERS.

SARS-CoV-1 and SARS-CoV-2 can cause severe respiratory disease in humans. The viral spike protein expressed by these viruses binds to angiotensin-converting enzyme 2 (ACE2). In some embodiments, the invention provides methods of treating a human suspected of having an infection with a Betacoronavirus causing SARS. In particular embodiments, the Betacoronavirus expresses a spike glycoprotein (S protein) that binds to ACE2, specifically human ACE2.

In a preferred embodiment of the invention, the infection is SARS-CoV-2.

SARS-CoV-1

In some embodiments, the invention provides for methods of treating a human suspected of having an infection with SARS-CoV-1 (severe acute respiratory syndrome coronavirus 1). SARS-CoV-1 is a positive-sense single-stranded RNA virus of the Betacoronavirus genus that causes SARS. In some embodiments, the invention provides for methods of treating a human having been diagnosed with SARS. The full genome sequences of various isolates from infected human patients are available from GenBank. The reference genome has the NCBI Reference Sequence ID NC_004718.

SARS-CoV-2

In some embodiments, the invention provides for methods of treating a human suspected of having an infection with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). SARS-CoV-2 (synonyms: 2019-nCoV, HCoV-19) is a positive-sense single-stranded RNA virus of the Betacoronavirus genus that causes the coronavirus disease termed COVTD-19. In some embodiments, the invention provides for methods of treating a human having been diagnosed with COVTD-19. The full genome sequences of various isolates from infected human patients are available from GenBank. The reference genome has the GenBank ID MN908947 (NCBI Reference Sequence ID NC_045512).

Symptoms of infection with SARS-CoV-2 may include, but are not limited to fever, chest pains, dry cough, dyspnea, headache, and hypoxemia. A human may be infected with SARS- CoV-2 but, at least initially, not demonstrate any symptoms of COVID-19. The invention encompasses the treatment of these patients.

Identifying subjects in need of treatment

In certain embodiments, the methods of treatment described herein may include steps for identifying a human suspected of having an infection with a coronavirus causing severe respiratory disease. Such identification steps are typically highly specific for a particular virus. For example, they may include testing for the presence of specific viruses known to cause respiratory disease, including coronaviruses such as MERS, SARS-CoV-1 and SARS-CoV-2. In some embodiments, testing includes step for specifically identifying SARS-CoV-1 and SARS- CoV-2. In a particular embodiment, a test for use with the invention is specific for SARS-CoV-2.

Identification may include detecting symptoms of the virus infection, and detecting virus- specific antigens, antibodies or nucleic acids in a biological sample. The term “biological sample” and used herein may include cell culture or extracts thereof; biopsied material obtained from a human; and blood, mucus, saliva, urine, feces, semen, tears or other body fluids or extracts thereof. In particular embodiments, steps for identifying humans suspected of having an infection with a coronavirus causing SARS may include real-time reverse transcription polymerase chain reaction (rRT-PCR). rRT-PCR may include detection of gene sequence specific to SARS-CoV-2, for example, those found in RdRP, ORFlab E and/or N genes. In some embodiments, multiple (e.g., 2 or 3) specific sequences in the same gene (e.g., the RdRP or N gene) are detected.

Therapeutic methods

As used herein, the term “treating” and its variants (e.g., “treat”, “treatment” etc.) is understood as the management and care of a patient for the purpose of combatting the disease, condition or disorder, including amelioration of one or more symptoms of a disease, condition or disorder.

As used herein, the term “prevent” and its variants (e.g., “preventing”) refers to the ability of a drug, or a combination of drugs, to stave off the occurrence of a clinically undesirable disease, disorder, symptom, or condition for a clinically significant period of time.

The term “therapeutically effective amount” used herein refers to the amount of anti- TNFa agent needed to treat or ameliorate the virus infection defined herein. The term “prophylactically effective amount” used herein refers to the amount of anti-TNFa agent needed to prevent the virus infection described herein. The exact dosage will generally be dependent on the patient’s status at the time of administration. Factors that may be taken into consideration when determining dosage include the severity of the virus state in the human, the general health of the human, the age, weight, gender, diet, time, frequency and route of administration, drug combinations, reaction sensitivities and the human’s tolerance or response to therapy. The precise amount can be determined by routine experimentation, but may ultimately lie with the judgement of the clinician. Particularly useful doses for use in this invention are set out below. The anti-TNFa agent may be administered individually to a human or may be administered in combination with one or more further agents (see “Combinations” section below). Alternatively, the anti-TNFa agent may be administered as a monotherapy.

Dosage regimen pharmaceutical form and modes of administration

Based on the preliminary findings reported, baseline blood TNFa levels are significantly elevated in severe and critical COVTD-19 patients. In another study, TNFa levels were found to correlate with disease severity with a substantial proportion of severe and critical COVID-19 patients having TNFa levels exceeding about 11 pg/mL. Of note, median TNFa levels in patients with active inflammatory bowel disease (IBD; including ulcerative colitis-UC and Crohn’s Disease— CD) ranged between 9.5 to 14.0 pg/mL. Accordingly, the TNFa levels seen in severe and critical COVID-19 patients appear to be in the range observed in patients with IBD. As the starting dose of infliximab in IBD is 5 mg/kg IV, this dose is hoped to provide sufficient exposure to bind both soluble and membrane bound TNFa efficiently in patients with confirmed severe or critical COVID-19 disease who are at risk of ARDS or other pulmonary diseases typically triggered by viral infection.

In a short-term study of patients with CD where single doses of 5 mg/kg, 10 mg/kg, or 20 mg/kg infliximab were evaluated, no apparent dose-response was observed for this range of doses with respect to either magnitude or duration of clinical response, and adverse events (AEs). Accordingly, it is hoped that doses greater than 5 mg/kg would not be needed for acute treatment of COVID-19, but the clinical data will be required to confirm that hypothesis. In addition, with respect to treatment duration, because the clinical course of the disease lasts about 3 weeks, our hypothesis is that a single dose of 5 mg/kg infliximab should provide adequate exposure to suppress elevated TNFa levels for at least 3 weeks. The clinical data will be required to confirm that hypothesis. In summary, infliximab 5 mg/kg administered as a single IV infusion is hoped to be effective with acceptable risks in rapidly suppressing the elevated TNFa levels in participants with confirmed severe or critical COVID-19 disease as no efficacy data are available on the use of infliximab to treat COVID-19 induced ARDS..

Antibodies and Related Properties The antibodies of the invention can bind human TNFa with a wide range of affinities (KD). In a preferred embodiment, at least one human mAh of the present invention can optionally bind human TNFa with high affinity. For example, a human or human engineered mAh can bind human TNFa with a KD equal to or less than about 10⁷ M, such as but not limited to, 0.1-9.9 (or any range or value therein) x 10⁷, 10⁸, 10⁹, 10 ¹⁰, 10 ¹¹, 10 ¹², 10 ¹³, 10 ¹⁴, 10 ¹⁵ or any range or value therein, as determined by surface plasmon resonance or the Kinexa method, as practiced by those of skill in the art.

The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method. (See, for example, Berzofsky, et al., “Antibody- Antigen Interactions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York, NY (1992); and methods described herein). The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD, K_on, K_0ff) are preferably made with standardized solutions of antibody and antigen, and a standardized buffer, such as the buffer described herein.

A preferred anti-TNFa antibody of the invention is Remicade® (infliximab). In accordance with the present invention, the anti-TNFa antibody comprises an antibody in which the light and heavy chain variable regions (SEQ ID NO:s 1 and 2 respectively) or CDRs incorporated therein, are derived from infliximab and bind to and inhibit the function of human TNFa, and the framework and constant regions of the antibody are derived from one or more human antibodies. The variable region or CDRs derived from infliximab preferably have from about 90% to about 100% identity with the variable region or CDRs of infliximab, although any and all modifications, including substitutions, insertions and deletions, are contemplated so long as the chimeric antibody maintains the ability to bind to and inhibit TNFa. The regions of the chimeric, humanized or CDR-grafted antibodies that are derived from human antibodies need not have 100% identity with the human antibodies. In a preferred embodiment, as many of the human amino acid residues as possible are retained in order than immunogenicity is negligible, but the human residues, in particular residues of the framework region, are substituted as required and as taught herein below in accordance with the present invention. Such modifications as disclosed herein are necessary to support the antigen binding site formed by the CDRs while simultaneously maximizing the humanization of the antibody. An anti-TNFa antibody according to the present invention includes any protein or peptide containing a molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to, at least one ligand binding portion (LBP), such as but not limited to, a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a framework region (e.g., FR1, FR2, FR3, FR4 or fragment thereof, further optionally comprising at least one substitution, insertion or deletion), a heavy chain or light chain constant region, (e.g., comprising at least one CHI, hinge 1, hinge2, hinge3, hinge4, CH2, or CH3 or fragment thereof, further optionally comprising at least one substitution, insertion or deletion), or any portion thereof, that can be incorporated into an antibody of the present invention. An antibody of the invention can include or be derived from any mammal, such as but not limited to, a human, a mouse, a rabbit, a rat, a rodent, a primate, or any combination thereof, and the like.

The isolated antibodies of the present invention comprise the antibody amino acid sequences disclosed herein encoded by any suitable polynucleotide, or any isolated or prepared antibody. Preferably, the human antibody or antigen-binding fragment binds human TNFa and, thereby, partially or substantially neutralizes at least one biological activity of the protein. An antibody, or specified portion or variant thereof, that partially or preferably substantially neutralizes at least one biological activity of at least one TNFa protein or fragment can bind the protein or fragment and thereby inhibit activities mediated through the binding of TNFa to the TNFa receptor or through other TNFa-dependent or mediated mechanisms. As used herein, the term “neutralizing antibody” refers to an antibody that can inhibit an TNFa-dependent activity by about 20-120%, preferably by at least about 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or more depending on the assay. The capacity of an anti- TNFa antibody to inhibit an TNFa-dependent activity is preferably assessed by at least one suitable TNFa protein or receptor assay, as described herein and/or as known in the art. A human antibody of the invention can be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa or lambda light chain. In one embodiment, the human antibody comprises an IgG heavy chain or defined fragment, for example, at least one of isotypes, IgGl , IgG2, IgG3 or IgG4. Antibodies of this type can be prepared by employing a transgenic mouse or other transgenic non-human mammal comprising at least one human light chain (e.g., IgG, IgA, and IgM) transgenes as described herein and/or as known in the art. In another embodiment, the anti-human TNFa human antibody comprises an IgGl heavy chain and an IgGl light chain. Generally, the human antibody or antigen-binding fragment of the present invention will comprise an antigen-binding region that comprises at least one human complementarity determining region (CDR1, CDR2 and CDR3) or variant of at least one heavy chain variable region and at least one human complementarity determining region (CDR1, CDR2 and CDR3) or variant of at least one light chain variable region. The CDR sequences may be derived from human germline sequences or closely match the germline sequences. For example, the CDRs from a synthetic library derived from the original mouse CDRs can be used. These CDRs may be formed by incorporation of conservative substitutions from the original mouse sequence.

At least one antibody of the present invention can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of an antibody to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to an antibody of the present invention to facilitate purification. Such regions can be removed prior to final preparation of an antibody or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

Illustrative of cell cultures useful for the production of the antibodies, specified portions or variants thereof, are mammalian cells. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions or bioreactors can also be used. A number of suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, and include the COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL- 1651), HEK293, BHK21 (e.g, ATCC CRL- 10), CHO (e.g, ATCC CRL 1610) and BSC-1 (e.g, ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0-Agl4, 293 cells, HeLa cells and the like, which are readily available from, for example, American Type Culture Collection, Manassas, Va (www.atcc.org). Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Agl4 cells (ATCC Accession Number CRL- 1851). In a particularly preferred embodiment, the recombinant cell is a P3X63Ab8.653 or a SP2/0-Agl4 cell.

Expression vectors for these cells can include one or more of the following expression control sequences, such as, but not limited to, an origin of replication; a promoter (e.g., late or early SV40 promoters, the CMV promoter (US Pat.Nos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (US Pat.No. 5,266,491), at least one human immunoglobulin promoter; an enhancer, and/or processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra. Other cells useful for production of nucleic acids or proteins of the present invention are known and/or available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources.

Purification of an Antibody

An anti-TNFa antibody can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography ("HPLC") can also be employed for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein by reference.

Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the antibody of the present invention can be glycosylated or can be non-glycosylated, with glycosylated preferred. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein Science, supra, Chapters 12-14, all entirely incorporated herein by reference.

Amino Acid Codes

The amino acids that make up anti-TNFa antibodies of the present invention are often abbreviated. The amino acid designations can be indicated by designating the amino acid by its single letter code, its three letter code, name, or three nucleotide codon(s) as is well understood in the art (see Alberts, B., et al, Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994) An anti-TNFa antibody of the present invention can include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation, as specified herein. Amino acids in an anti-TNFa antibody of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least one TNFa neutralizing activity. Sites that are critical for antibody binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al, J. Mol. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).

Non-limiting variants that can enhance or maintain at least one of the listed activities include, but are not limited to, any of the above polypeptides, further comprising at least one mutation corresponding to at least one substitution in the residues varied among the disclosed variant amino acid sequences.

In another aspect, the invention relates to human antibodies and antigen-binding fragments, as described herein, which are modified by the covalent attachment of an organic moiety. Such modification can produce an antibody or antigen-binding fragment with improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic moiety can be a linear or branched hydrophilic polymeric group, fatty acid group, or fatty acid ester group. In particular embodiments, the hydrophilic polymeric group can have a molecular weight of about 800 to about 120,000 Daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid ester group can comprise from about eight to about forty carbon atoms.

The modified antibodies and antigen-binding fragments of the invention can comprise one or more organic moieties that are covalently bonded, directly or indirectly, to the antibody. Each organic moiety that is bonded to an antibody or antigen-binding fragment of the invention can independently be a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group. As used herein, the term “fatty acid” encompasses mono-carboxylic acids and di- carboxylic acids. A “hydrophilic polymeric group,” as the term is used herein, refers to an organic polymer that is more soluble in water than in octane. For example, poly lysine is more soluble in water than in octane. Thus, an antibody modified by the covalent attachment of polylysine is encompassed by the invention. Hydrophilic polymers suitable for modifying antibodies of the invention can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl pyrolidone. Preferably, the hydrophilic polymer that modifies the antibody of the invention has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity. For example, PEG5000 and PEG20,000, wherein the subscript is the average molecular weight of the polymer in Daltons, can be used. The hydrophilic polymeric group can be substituted with one to about six alkyl, fatty acid or fatty acid ester groups. Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be prepared by employing suitable methods. For example, a polymer comprising an amine group can be coupled to a carboxylate of the fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with N, N-carbonyl diimidazole) on a fatty acid or fatty acid ester can be coupled to a hydroxyl group on a polymer.

Fatty acids and fatty acid esters suitable for modifying antibodies of the invention can be saturated or can contain one or more units of unsaturation. Fatty acids that are suitable for modifying antibodies of the invention include, for example, n-dodecanoate (Cl 2, laurate), n- tetradecanoate (Cl 4, myristate), n-octadecanoate (Cl 8, stearate), n-eicosanoate (C20, arachidate) , n-docosanoate (C22, behenate), n-triacontanoate (C30), n-tetracontanoate (C40), cis-9- octadecanoate (C18, oleate), all cis-5,8,ll,14-eicosatetraenoate (C20, arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include mono-esters of dicarboxylic acids that comprise a linear or branched lower alkyl group. The lower alkyl group can comprise from one to about twelve, preferably, one to about six, carbon atoms.

The modified human antibodies and antigen-binding fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents. A “modifying agent” as the term is used herein, refers to a suitable organic group (e.g., hydrophilic polymer, a fatty acid, a fatty acid ester) that comprises an activating group. An "activating group" is a chemical moiety or functional group that can, under appropriate conditions, react with a second chemical group thereby forming a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups, such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehyde functional group can be coupled to amine- or hydrazide-containing molecules, and an azide group can react with a trivalent phosphorous group to form phosphoramidate or phosphorimide linkages. Suitable methods to introduce activating groups into molecules are known in the art (see for example, Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996)). An activating group can be bonded directly to the organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester), or through a linker moiety, for example, a divalent Cl -Cl 2 group wherein one or more carbon atoms can be replaced by a heteroatom, such as oxygen, nitrogen or sulfur. Suitable linker moieties include, for example, tetraethylene glycol, -(CH2)3-, -NH-(CH2)6-NH-, -(CH2)2-NH- and -CH2-0-CH2-CH2-0-CH2-CH2-0-CH-NH-. Modifying agents that comprise a linker moiety can be produced, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate. The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate, as described, or can be reacted with maleic anhydride and the resulting product cyclized to produce an activated maleimido derivative of the fatty acid. (See, for example, Thompson, et al., WO 92/16221, the entire teachings of which are incorporated herein by reference.)

The modified antibodies of the invention can be produced by reacting a human antibody or antigen-binding fragment with a modifying agent. For example, the organic moieties can be bonded to the antibody in a non-site specific manner by employing an amine-reactive modifying agent, for example, an NHS ester of PEG. Modified human antibodies or antigen-binding fragments can also be prepared by reducing disulfide bonds (e.g., intra-chain disulfide bonds) of an antibody or antigen-binding fragment. The reduced antibody or antigen-binding fragment can then be reacted with a thiol-reactive modifying agent to produce the modified antibody of the invention. Modified human antibodies and antigen-binding fragments comprising an organic moiety that is bonded to specific sites of an antibody of the present invention can be prepared using suitable methods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3: 147-153 (1992); Werlen et al, Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci.

6(10): 2233 -2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and the methods described in Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996).

Anti-TNFa antibody compounds, compositions or combinations of the present invention can further comprise at least one of any suitable auxiliary, such as, but not limited to, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. Pharmaceutically acceptable auxiliaries are preferred. Non-limiting examples of, and methods of preparing such sterile solutions are well known in the art, such as, but limited to, Gennaro, Ed., Remington’s Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, PA) 1990. Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and/or stability of the anti-TNFa antibody, fragment or variant composition as well known in the art or as described herein.

Pharmaceutical excipients and additives useful in the present composition include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, -tri-, tetra-, and oligosaccharides; derivatized sugars, such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1 -99.99% by weight or volume. Exemplary protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody components, which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

Carbohydrate excipients suitable for use in the composition of the invention include, for example, monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myoinositol and the like. Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose, and raffinose.

Anti-TNFa antibody compositions can also include a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. Preferred buffers for use in the present compositions are organic acid salts, such as citrate.

Additionally, anti-TNFa antibody compositions of the invention can include polymeric excipients/additives, such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-P-cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates, such as “TWEEN 20” and “TWEEN 80”), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

These and additional known pharmaceutical excipients and/or additives suitable for use in the anti-TNFa antibody, portion or variant compositions according to the invention are known in the art, e.g., as listed in “Remington: The Science & Practice of Pharmacy”, 19th ed., Williams & Williams, (1995), and in the “Physician’s Desk Reference”, 52nd ed., Medical Economics, Montvale, NJ (1998), the disclosures of which are entirely incorporated herein by reference. Preferred carrier or excipient materials are carbohydrates (e.g., saccharides and alditols) and buffers (e.g., citrate) or polymeric agents. An exemplary carrier molecule is the mucopolysaccharide, hyaluronic acid, which may be useful for intraarticular delivery. Formulations

As noted above, the invention provides for stable formulations, which preferably comprise a phosphate buffer with saline or a chosen salt, as well as preserved solutions and formulations containing a preservative as well as multi-use preserved formulations suitable for pharmaceutical or veterinary use, comprising at least one anti-TNFa antibody in a pharmaceutically acceptable formulation. Preserved formulations contain at least one known preservative or optionally selected from the group consisting of at least one phenol, m-cresol, p- cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, polymers, or mixtures thereof in an aqueous diluent. Any suitable concentration or mixture can be used as known in the art, such as about 0.0015%, or any range, value, or fraction therein. Non-limiting examples include, no preservative, about 0.1-2% m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), about 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), about 0.001-0.5% thimerosal (e.g., 0.005, 0.01), about 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

As noted above, the invention provides an article of manufacture, comprising packaging material and at least one vial comprising a solution of at least one anti-TNFa antibody with the prescribed buffers and/or preservatives, optionally in an aqueous diluent, wherein said packaging material comprises a label that indicates that such solution can be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or greater. The invention further comprises an article of manufacture, comprising packaging material, a first vial comprising lyophilized at least one anti-TNFa antibody, and a second vial comprising an aqueous diluent of prescribed buffer or preservative, wherein said packaging material comprises a label that instructs a patient to reconstitute the at least one anti-TNFa antibody in the aqueous diluent to form a solution that can be held over a period of twenty-four hours or greater.

The at least one anti-TNFa antibody used in accordance with the present invention can be produced by recombinant means, including from mammalian cell or transgenic preparations, or can be purified from other biological sources, as described herein or as known in the art.

The range of at least one anti-TNFa antibody in the use and method of the present invention includes amounts yielding upon reconstitution, if in a wet/dry system, concentrations from about 1.0 pg/ml to about 1000 mg/ml, although lower and higher concentrations are operable and are dependent on the intended delivery vehicle, e.g., solution formulations will differ from transdermal patch, pulmonary, transmucosal, or osmotic or micro pump methods.

Preferably, the aqueous diluent optionally further comprises a pharmaceutically acceptable preservative. Preferred preservatives include those selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof. The concentration of preservative used in the formulation is a concentration sufficient to yield an anti-microbial effect. Such concentrations are dependent on the preservative selected and are readily determined by the skilled artisan.

Other excipients, e.g., isotonicity agents, buffers, antioxidants, and preservative enhancers, can be optionally and preferably added to the diluent. An isotonicity agent, such as glycerin, is commonly used at known concentrations. A physiologically tolerated buffer is preferably added to provide improved pH control. The formulations can cover a wide range of pHs, such as from about pH 4 to about pH 10, and preferred ranges from about pH 5 to about pH 9, and a most preferred range of about 6.0 to about 8.0. Preferably, the formulations of the present invention have a pH between about 6.8 and about 7.8. Preferred buffers include phosphate buffers, most preferably, sodium phosphate, particularly, phosphate buffered saline (PBS).

Other additives, such as a pharmaceutically acceptable solubilizers like Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymers), and PEG (polyethylene glycol) or non ionic surfactants, such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic® polyls, other block co-polymers, and chelators, such as EDTA and EGTA, can optionally be added to the formulations or compositions to reduce aggregation. These additives are particularly useful if a pump or plastic container is used to administer the formulation. The presence of pharmaceutically acceptable surfactant mitigates the propensity for the protein to aggregate.

The formulations of the present invention can be prepared by a process which comprises mixing at least one anti-TNFa antibody and a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof in an aqueous diluent. Mixing the at least one anti-TNFa antibody and preservative in an aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of at least one anti-TNFa antibody in buffered solution is combined with the desired preservative in a buffered solution in quantities sufficient to provide the protein and preservative at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used.

The formulations according to the invention can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized at least one anti-TNFa antibody that is reconstituted with a second vial containing water, a preservative and/or excipients, preferably, a phosphate buffer and/or saline and a chosen salt, in an aqueous diluent. Either a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus can provide a more convenient treatment regimen than currently available.

The present claimed products are useful for administration over a period ranging from immediate to twenty-four hours or greater. Accordingly, the presently claimed articles of manufacture offer significant advantages to the patient. Formulations of the invention can optionally be safely stored at temperatures of from about 2°C to about 40°C and retain the biological activity of the antibody for extended periods of time, thus allowing a package label indicating that the solution can be held and/or used over a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or greater. If preserved diluent is used, such label can include use up to 1-12 months, one-half, one and a half, and/or two years.

The solutions of at least one anti-TNFa antibody of the invention can be prepared by a process that comprises mixing at least one antibody in an aqueous diluent. Mixing is carried out using conventional dissolution and mixing procedures. To prepare a suitable diluent, for example, a measured amount of at least one antibody in water or buffer is combined in quantities sufficient to provide the protein and, optionally, a preservative or buffer at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used.

The antibody can be provided to patients as clear solutions or as vials comprising a vial of lyophilized anti-TNFa antibody that is reconstituted with a second vial containing the aqueous diluent. Either a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus provides a more convenient treatment regimen than currently available.

The antibody can be provided indirectly to patients by providing to pharmacies, clinics, or other such institutions and facilities, clear solutions or dual vials comprising a vial of lyophilized anti-TNFa antibody that is reconstituted with a second vial containing the aqueous diluent. The clear solution in this case can be up to one liter or even larger in size, providing a large reservoir from which smaller portions of the antibody solution can be retrieved one or multiple times for transfer into smaller vials and provided by the pharmacy or clinic to their customers and/or patients. Recognized devices comprising single vial systems include pen-injector devices for delivery of a solution, such as BD Pens, BD Autojector®, Humaject®, NovoPen®, B-D®Pen, AutoPen®, and OptiPen®, GenotropinPen®, Genotronorm Pen®, Humatro Pen®, Reco-Pen®, Roferon Pen®, Biojector®, Iject®, J-tip Needle-Free Injector®, Intraject®, Medi-Ject®, e.g., as made or developed by Becton Dickensen (Franklin Lakes, NJ, www.bectondickenson.com), Disetronic (Burgdorf, Switzerland, www.disetronic.com; Bioject, Portland, Oregon (www.bioject.com); National Medical Products, Weston Medical (Peterborough, UK, www.weston-medical.com), Medi-Ject Corp (Minneapolis, MN, www. mediject.com), and similarly suitable devices, such as OnePress™. Recognized devices comprising a dual vial system include those pen-injector systems for reconstituting a lyophilized drug in a cartridge for delivery of the reconstituted solution, such as the HumatroPen®. Examples of other devices suitable include pre-filled syringes, auto-injectors, needle free injectors and needle free IV infusion sets.

The products used in the presently claimed uses and methods include packaging material. The packaging material provides, in addition to the information required by the regulatory agencies, the conditions under which the product can be used. The packaging material provides instructions to the patient to reconstitute the anti-TNFa antibody in the aqueous diluent to form a solution and to use the solution over a period of 2-24 hours or greater for the two vial, wet/dry, product. For the single vial, solution product, the label indicates that such solution can be used over a period of 2-24 hours or greater. The products are useful for human pharmaceutical product use.

The formulations used in the present invention can be prepared by a process that comprises mixing an anti-TNFa antibody and a selected buffer, preferably, a phosphate buffer containing saline or a chosen salt. Mixing the anti-TNFa antibody and buffer in an aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of at least one antibody in water or buffer is combined with the desired buffering agent in water in quantities sufficient to provide the protein and buffer at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used. The claimed stable or preserved formulations can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized an anti-TNFa antibody that is reconstituted with a second vial containing a preservative or buffer and excipients in an aqueous diluent. Either a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus provides a more convenient treatment regimen than currently available.

Other formulations or methods of stabilizing the anti-TNFa antibody may result in other than a clear solution of lyophilized powder comprising the antibody. Among non-clear solutions are formulations comprising particulate suspensions, said particulates being a composition containing the anti-TNFa antibody in a structure of variable dimension and known variously as a microsphere, microparticle, nanoparticle, nanosphere, or liposome. Such relatively homogenous, essentially spherical, particulate formulations containing an active agent can be formed by contacting an aqueous phase containing the active agent and a polymer and a nonaqueous phase followed by evaporation of the nonaqueous phase to cause the coalescence of particles from the aqueous phase as taught in U.S. 4,589,330. Porous microparticles can be prepared using a first phase containing active agent and a polymer dispersed in a continuous solvent and removing said solvent from the suspension by freeze-drying or dilution-extraction-precipitation as taught in FT.S. 4,818,542. Preferred polymers for such preparations are natural or synthetic copolymers or polymers selected from the group consisting of gelatin agar, starch, arabinogalactan, albumin, collagen, polyglycolic acid, polylactic aced, glycolide-F(-) lactide poly(episilon-caprolactone, poly(epsilon-caprolactone-CO-lactic acid), poly(epsilon-caprolactone-CO-glycolic acid), poly(B- hydroxy butyric acid), polyethylene oxide, polyethylene, poly(alkyl-2-cyanoacrylate), poly(hydroxyethyl methacrylate), polyamides, poly(amino acids), poly(2-hydroxyethyl DF- aspartamide), poly(ester urea), poly(F-phenylalanine/ethylene glycol/1, 6-diisocyanatohexane) and poly(methyl methacrylate). Particularly preferred polymers are polyesters, such as polyglycolic acid, polylactic aced, glycolide-F(-) lactide poly(episilon-caprolactone, poly(epsilon-caprolactone-CO-lactic acid), and poly(epsilon-caprolactone-CO-gly colic acid. Solvents useful for dissolving the polymer and/or the active include: water, hexafluoroisopropanol, methylenechloride, tetrahydrofuran, hexane, benzene, or hexafluoroacetone sesquihydrate. The process of dispersing the active containing phase with a second phase may include pressure forcing said first phase through an orifice in a nozzle to affect droplet formation. Dry powder formulations may result from processes other than lyophilization, such as by spray drying or solvent extraction by evaporation or by precipitation of a crystalline composition followed by one or more steps to remove aqueous or nonaqueous solvent. Preparation of a spray-dried antibody preparation is taught in U.S. 6,019,968. The antibody-based dry powder compositions may be produced by spray drying solutions or slurries of the antibody and, optionally, excipients, in a solvent under conditions to provide a respirable dry powder. Solvents may include polar compounds, such as water and ethanol, which may be readily dried. Antibody stability may be enhanced by performing the spray drying procedures in the absence of oxygen, such as under a nitrogen blanket or by using nitrogen as the drying gas. Another relatively dry formulation is a dispersion of a plurality of perforated microstructures dispersed in a suspension medium that typically comprises a hydrofluoroalkane propellant as taught in WO 9916419. The stabilized dispersions may be administered to the lung of a patient using a metered dose inhaler. Equipment useful in the commercial manufacture of spray dried medicaments are manufactured by Buchi Ltd. or Niro Corp.

An anti-TNFa antibody in either the stable or preserved formulations or solutions described herein, can be administered to a patient in accordance with the present invention via a variety of delivery methods including SC or IM injection; transdermal, pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump, or other means appreciated by the skilled artisan, as well-known in the art.

Alternative Administration

Many known and developed modes can be used according to the present invention for administering pharmaceutically effective amounts of an anti-TNFa antibody according to the present invention. While pulmonary administration is used in the following description, other modes of administration can be used according to the present invention with suitable results. TNFa antibodies of the present invention can be delivered in a carrier, as a solution, emulsion, colloid, or suspension, or as a dry powder, using any of a variety of devices and methods suitable for administration by inhalation or other modes described here within or known in the art. Parenteral Formulations and Administration

Formulations for parenteral administration can contain as common excipients sterile water or saline, polyalkylene glycols, such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. Aqueous or oily suspensions for injection can be prepared by using an appropriate emulsifier or humidifier and a suspending agent, according to known methods. Agents for injection can be a non-toxic, non-orally administrable diluting agent, such as aqueous solution, a sterile injectable solution or suspension in a solvent. As the usable vehicle or solvent, water, Ringer's solution, isotonic saline, etc. are allowed; as an ordinary solvent or suspending solvent, sterile involatile oil can be used. For these purposes, any kind of involatile oil and fatty acid can be used, including natural or synthetic or semisynthetic fatty oils or fatty acids; natural or synthetic or semisynthetic mono- or di- or tri-glycerides. Parental administration is known in the art and includes, but is not limited to, conventional means of injections, a gas pressured needle-less injection device as described in U.S. Pat. No. 5,851,198, and a laser perforator device as described in U.S. Pat. No. 5,839,446 entirely incorporated herein by reference.

Alternative Delivery

The invention further relates to the administration of an anti-TNFa antibody by parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal means. An anti-TNFa antibody composition can be prepared for use for parenteral (subcutaneous, intramuscular or intravenous) or any other administration particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration particularly in semisolid forms, such as, but not limited to, creams and suppositories; for buccal, or sublingual administration, such as, but not limited to, in the form of tablets or capsules; or intranasally, such as, but not limited to, the form of powders, nasal drops or aerosols or certain agents; or transdermally, such as not limited to a gel, ointment, lotion, suspension or patch delivery system with chemical enhancers such as dimethyl sulfoxide to either modify the skin structure or to increase the drug concentration in the transdermal patch (Junginger, et al. In "Drug Permeation Enhancement;" Hsieh, D. S., Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994, entirely incorporated herein by reference), or with oxidizing agents that enable the application of formulations containing proteins and peptides onto the skin (WO 98/53847), or applications of electric fields to create transient transport pathways, such as electroporation, or to increase the mobility of charged drugs through the skin, such as iontophoresis, or application of ultrasound, such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the above publications and patents being entirely incorporated herein by reference). Pulmonary/Nasal Administration

For pulmonary administration, preferably, an anti-TNFa antibody composition is delivered in a particle size effective for reaching the lower airways of the lung or sinuses. According to the invention, an anti-TNFa antibody can be delivered by any of a variety of inhalation or nasal devices known in the art for administration of a therapeutic agent by inhalation. These devices capable of depositing aerosolized formulations in the sinus cavity or alveoli of a patient include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Other devices suitable for directing the pulmonary or nasal administration of antibodies are also known in the art. All such devices can use formulations suitable for the administration for the dispensing of antibody in an aerosol. Such aerosols can be comprised of either solutions (both aqueous and non-aqueous) or solid particles.

Metered dose inhalers like the Ventolin® metered dose inhaler, typically use a propellent gas and require actuation during inspiration (See, e.g., WO 94/16970, WO 98/35888). Dry powder inhalers like TurbuhalerTM (Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), SpirosTM inhaler (Dura), devices marketed by Inhale Therapeutics, and the Spinhaler® powder inhaler (Fisons), use breath-actuation of a mixed powder (US 4668218 Astra, EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, US 5458135 Inhale, WO 94/06498 Fisons, entirely incorporated herein by reference). Nebulizers like AERxTM Aradigm, the Ultravent® nebulizer (Mallinckrodt), and the Acorn II® nebulizer (Marquest Medical Products) (US 5404871 Aradigm, WO 97/22376), the above references entirely incorporated herein by reference, produce aerosols from solutions, while metered dose inhalers, dry powder inhalers, etc. generate small particle aerosols. These specific examples of commercially available inhalation devices are intended to be a representative of specific devices suitable for the practice of this invention, and are not intended as limiting the scope of the invention.

Preferably, a composition comprising an anti-TNFa antibody is delivered by a dry powder inhaler or a sprayer. There are several desirable features of an inhalation device for administering an antibody of the present invention. For example, delivery by the inhalation device is advantageously reliable, reproducible, and accurate. The inhalation device can optionally deliver small dry particles, e.g., less than about 10 pm, preferably about 1-5 pm, for good respirability. Administration of TNFa Antibody Compositions as a Spray

A spray including TNFa antibody composition can be produced by forcing a suspension or solution of an anti-TNFa antibody through a nozzle under pressure. The nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size. An electrospray can be produced, for example, by an electric field in connection with a capillary or nozzle feed. Advantageously, particles of an anti-TNFa antibody composition delivered by a sprayer have a particle size less than about 10 pm, preferably, in the range of about 1 pm to about 5 pm, and, most preferably, about 2 pm to about 3 pm.

Formulations of an anti-TNFa antibody composition suitable for use with a sprayer typically include antibody composition in an aqueous solution at a concentration of about 0.1 mg to about 100 mg of an anti-TNFa antibody composition per ml of solution or mg/gm, or any range, value, or fraction therein. The formulation can include agents, such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc. The formulation can also include an excipient or agent for stabilization of the antibody composition, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating antibody compositions include albumin, protamine, or the like. Typical carbohydrates useful in formulating antibody compositions include sucrose, mannitol, lactose, trehalose, glucose, or the like. The antibody composition formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the antibody composition caused by atomization of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between 0.001 and 14% by weight of the formulation. Especially preferred surfactants for purposes of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for formulation of a protein, such as TNFa antibodies, or specified portions or variants, can also be included in the formulation.

Oral Formulations and Administration

Formulations for oral administration rely on the co-administration of adjuvants (e.g., resorcinols and nonionic surfactants, such as polyoxyethylene oleyl ether and n- hexadecylpolyethylene ether) to increase artificially the permeability of the intestinal walls, as well as the co-administration of enzymatic inhibitors (e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymatic degradation. Formulations for delivery of hydrophilic agents including proteins and antibodies and a combination of at least two surfactants intended for oral, buccal, mucosal, nasal, pulmonary, vaginal transmembrane, or rectal administration are taught in U.S. 6,309,663. The active constituent compound of the solid- type dosage form for oral administration can be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starches, agar, arginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, and glyceride. These dosage forms can also contain other type(s) of additives, e.g., inactive diluting agent, lubricant, such as magnesium stearate, paraben, preserving agent, such as sorbic acid, ascorbic acid, .alpha. -tocopherol, antioxidant such as cysteine, disintegrator, binder, thickener, buffering agent, sweetening agent, flavoring agent, perfuming agent, etc.

Tablets and pills can be further processed into enteric-coated preparations. The liquid preparations for oral administration include emulsion, syrup, elixir, suspension and solution preparations allowable for medical use. These preparations can contain inactive diluting agents ordinarily used in said field, e.g., water. Liposomes have also been described as drug delivery systems for insulin and heparin (U.S. Pat. No. 4,239,754). More recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used to deliver pharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carrier compounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No. 5,5,871,753 and used to deliver biologically active agents orally are known in the art.

Mucosal Formulations and Administration

A formulation for orally administering a bioactive agent encapsulated in one or more biocompatible polymer or copolymer excipients, preferably, a biodegradable polymer or copolymer, affording microcapsules which due to the proper size of the resultant microcapsules results in the agent reaching and being taken up by the folliculi lymphatic aggregati, otherwise known as the "Peyer's patch," or "GALT" of the animal without loss of effectiveness due to the agent having passed through the gastrointestinal tract. Similar folliculi lymphatic aggregati can be found in the bronchei tubes (BALT) and the large intestine. The above-described tissues are referred to in general as mucosally associated lymphoreticular tissues (MALT). For absorption through mucosal surfaces, compositions and methods of administering at least one anti-TNFa antibody include an emulsion comprising a plurality of submicron particles, a mucoadhesive macromolecule, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. Pat. No. 5,514,670). Mucous surfaces suitable for application of the emulsions of the present invention can include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, stomachic, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, e.g., suppositories, can contain as excipients, for example, polyalkyleneglycols, vaseline, cocoa butter, and the like. Formulations for intranasal administration can be solid and contain as excipients, for example, lactose or can be aqueous or oily solutions of nasal drops.

For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelinatined starch, and the like (U.S. Pat. No. 5,849,695).

Transdermal Formulations and Administration

For transdermal administration, the anti-TNFa antibody is encapsulated in a delivery device, such as a liposome or polymeric nanoparticles, microparticle, microcapsule, or microspheres (referred to collectively as microparticles unless otherwise stated). A number of suitable devices are known, including microparticles made of synthetic polymers, such as polyhydroxy acids, such as polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers, such as collagen, polyamino acids, albumin and other proteins, alginate and other polysaccharides, and combinations thereof (U.S. Pat. No. 5,814,599).

Prolonged Administration and Formulations

It can be desirable to deliver the compounds of the present invention to the subject over prolonged periods of time, for example, for periods of one week to one year from a single administration. Various slow release, depot or implant dosage forms can be utilized. For example, a dosage form can contain a pharmaceutically acceptable non-toxic salt of the compounds that has a low degree of solubility in body fluids, for example, (a) an acid addition salt with a polybasic acid, such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) a salt with a polyvalent metal cation, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the like, or with an organic cation formed from e.g., N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of (a) and (b), e.g., a zinc tannate salt. Additionally, the compounds of the present invention or, preferably, a relatively insoluble salt, such as those just described, can be formulated in a gel, for example, an aluminum monostearate gel with, e.g., sesame oil, suitable for injection. Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, and the like. Another type of slow release depot formulation for injection would contain the compound or salt dispersed for encapsulation in a slow degrading, non-toxic, non-antigenic polymer, such as a polylactic acid/polyglycolic acid polymer for example as described in U.S. Pat. No. 3,773,919. The compounds or, preferably, relatively insoluble salts, such as those described above, can also be formulated in cholesterol matrix silastic pellets, particularly for use in animals. Additional slow release, depot or implant formulations, e.g., gas or liquid liposomes, are known in the literature (U.S. Pat. No. 5,770,222 and "Sustained and Controlled Release Drug Delivery Systems", J. R. Robinson ed., Marcel Dekker, Inc., N.Y., 1978).

Combinations

In one embodiment of the invention, the anti-TNFa antibody or agent is co-administered with one or more further agents. The further agent may be an antiviral (including antiretroviral) agent. In an embodiment, the antiviral agent is a small molecule. The small molecule may be independently selected from the group consisting of itraconozole, favipiravir (e.g., Avigan), remdesivir, ifenprodil, chloroquine, umifenovir (e.g., Arbidol), APNOl, galidesivir, ritonavir, BPI-002, OYA1, and SNG001. In an embodiment, the small molecule is favipiravir (e.g., Avigan). In an embodiment, the small molecule is remdesivir. In an embodiment, the small molecule is ifenprodil. In an embodiment, the small molecule is chloroquine. In an embodiment, the small molecule is umifenovir (e.g., Arbidol). In an embodiment, the small molecule is APNOl. In an embodiment, the small molecule is galidesivir. In an embodiment, the small molecule is ritonavir. In an embodiment, the small molecule is BPI-002. In an embodiment, the small molecule is OYA1. In an embodiment, the small molecule is SNG001.

In an embodiment, the antiviral agent is a vaccine. The vaccine may be independently selected from the group consisting of a MERS-CoV vaccine, an Infectious Bronchitis Virus (IBV) vaccine, an RNA vaccine, e.g., an mRNA vaccine such as mRNA-1273, a DNA vaccine such as INO-4700 (GLS-5300) or INO-4800, a subunit vaccine, a live vaccine such as TNX- 1800 and a recombinant vaccine. In one embodiment, the vaccine is co-administered with anti- TNFa agent for the prevention of infection of a human with SARS-CoV-1 and/or SARS-CoV-2.

In an embodiment, the antiviral agent is a protein. The protein may be a human recombinant protein such as AT-100 (rhSP-D). In another embodiment, the protein is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an antibody against human granulocyte-macrophage colony stimulating factor (GM-CSF). In some embodiments, the antibody is an antibody against human CCR5 receptor. In some embodiments, the antibody is antibody against a viral surface antigen, e.g., the spike protein (S protein). In an embodiment, the further agent is a plasma-derived agent such as TAK-888. In another embodiment, the further agent is an amniotic fluid concentrate. In another embodiment, the further agent is remestemcel-L (e.g., Ryoncil).

In an embodiment, the further agent alleviates one or more symptoms of an infection of a human with SARS-CoV-1 and/or SARS-CoV-2. Symptoms may include, but are not limited to fever, chest pains, dry cough, dyspnea, headache, and hypoxemia. By co-administered, it is meant simultaneous or sequential administration of anti-TNFa agent and one or more of the further agents defined herein. Simultaneous administration can mean administration of agent(s) in close proximity temporally, or administering a first agent, and then a second (or multiples) once the first agent has been onboarded to a subject. Sequential administration shall not be limited by time and any period of time can elapse between administrations. All administrations are readily optimized by those skilled in the art. By further agent it is meant an approved agent, an agent which is in development, or an agent which is said to be useful for the treatment or prevention of infection of a human with SARS-CoV-1 and/or SARS-CoV-2, whichever is being treated or prevented by anti- TNFa agent.

Each of the agents listed above may be specifically combined with an anti-TNFa agent in one of the combination therapies disclosed herein. Each of those combinations is herein specifically individualized.

Clinical Studies and Infliximab - Novel Protocols and Treatment Regiments

Over 17,500 subjects have been exposed to infliximab during over 100 interventional clinical studies conducted by Janssen; Merck, Sharp & Dohme; and Mitsubishi Tanabe Pharma Corporation. As of 23 August 2019, infliximab is currently being studied or has been studied in the following clinical indications: Crohn’s disease (including pediatric Crohn’s disease), RA (including JRA), UC (including pediatric UC), MS, sepsis, CHF, psoriasis, asthma, sarcoidosis, PsA, AS, COPD, cancer related cachexia, GCA, Behcet’s disease, and acute Kawasaki disease. The development of infliximab for the treatment of MS, JRA, cancer related cachexia, sepsis, COPD, sarcoidosis, asthma, GCA, and CHF has been discontinued. Clinical studies of infliximab have included formulation for IV delivery and/or formulation for SC or IM delivery; however, the development of the SC/IM formulation is no longer being pursued.

As stated above, no clinical efficacy data are currently available and no dosing regimens have been developed or tested for the use of infliximab to treat COVID-19 induced symptoms including, ARDS. The novel protocols and methodologies described herein provide a framework for assessing the efficacy of infliximab for treatment of COVID-19. The table below provides a summary of the clinical objectives and endpoints.

COVID-19 Clinical Trial Plan - Objectives And Endpoints

At the time of data analysis, additional endpoints may be considered for analysis in function of the evolution in scientific knowledge on COVID-19.

Hypothesis The primary hypothesis of this study is that infliximab in combination with standard of care (SOC) results in a statistically significant shorter time to improvement (defined as an improvement of at least 2 categories relative to Day 1 on the 6-point ordinal clinical recovery scale) versus placebo in combination with SOC, in participants with confirmed severe or critical COVID-19 disease. The analyses and protocols described herein help frame the analysis and clinical data to inform the conclusion as to whether anti-TNFa antibody is effective to reduce viral- induced symptoms associated with Covid-19.

OVERALL DESIGN

For ease of consideration, below is an overview of the protocol/study design, with a description of the annotations.

Schematic Overview of the Study

ORC {qlw) looking at safety”

COVID-19: coronavirus disease; EOT: end of trial, ICU: intensive care unit, IV: intravenous, PBO: placebo, qlw: every week, SOC: standard of care

* Randomization will be stratified by age (<65 and >65 years of age) and by use of invasive mechanical ventilation (Fi02< 6L/min, Fi02 > 6L/min and mechanical ventilation) at the time of randomization.

° The DRC will review the interim data as soon as the first 30 participants have been dosed and have had at least 7 days of follow-up. Additional interim analyses may be performed depending on the enrolment rate to guide further development and to support interactions with Health Authorities.

# Up to Day 28 or until hospital discharge or study discontinuation, whichever comes first. If a participant is discharged before Day 28, a phone call visit will be performed at Day 28.

§ SOC treatment (during study treatment and follow-phase) is determined by the investigator based on local practice and consists of supportive care. The protocols and study design of described herein include a randomized, double-blind, placebo-controlled, multicenter, interventional Phase 2 study in hospitalized participants with confirmed severe or critical COVID-19 disease. The details and parameters are described in detail herein. A target of approximately 270 participants will be randomly assigned in a 2: 1 ratio to receive 1 of the following 2 treatments: Treatment Arm: infliximab 5 mg/kg IV single dose infusion on Day 1 + SOC treatment Control Arm: placebo IV single dose infusion on Day 1 + SOC treatment. Randomization will be stratified by age (<65 and >65 years of age) and by use of invasive mechanical ventilation (yes/no) at the time of randomization. The study will include a Screening Phase (on Day -1), a ‘Day 1 to Day 28’ Phase and a Post Day 28 Phase (phone calls on Week 8 and Week 12). The entire study duration for each participant will be 12 Weeks with daily study assessments up to Day 28 or day of discharge (whichever comes first), and phone call assessments thereafter, ie, at Day 28 in case of discharge prior to Day 28, at Week 8 and Week 12. The post Day 28 assessment schedule will remain the same for participants still hospitalized after Day 28. The study is considered completed with the completion of the last study assessment (phone call assessment at Week 12) for the last participant in the study or the discontinuation of the last participant in the study, whichever comes last.

NUMBER OF PARTICIPANTS

A target of approximately 270 participants will be randomized. INTERVENTION GROUPS AND DURATION

Description of Interventions

DESCRIPTION OF INTERVENTIONS

The study intervention will be administered to the participant via an IV infusion using a normal saline IV bag with a total volume of 250 mL. Infliximab will be injected into the bag for use in the treatment arm, the volume of the bag will be adjusted so that the total bag volume remains 250 mL.

No substance will be injected in the bag for the placebo arm, and the 250 mL IV bag will be used as such.

An unblinded pharmacist or qualified staff member will prepare the IV bags before distribution to the clinic.

PREPARATION/HANDLING/STORAGE

Infliximab must be stored at controlled temperatures ranging from 36°F to 46°F (2°C to 8°C) and protected from light.

Infliximab 5 mg/kg will be administered to the participant via an IV infusion at a rate of not more than 100 mL/min over a time period of not less than 2 hours using a total volume of 250 mL. Placebo participants will receive the IV infusion of 250 mL normal saline at the same rate. Aseptic procedures must be used during the study agent infusion.

Refer to the IPPI for additional guidance on study intervention preparation, handling, and storage.

EFFICACY EVALUATIONS Efficacy assessments will include all information necessary for the 6-point ordinal clinical recovery scale, level of consciousness (Glasgow coma score (GCS)), virology assessment, supplemental oxygen use, resting Sp02, and pulmonary X-ray. Details for selected assessments are provided below. SAFETY EVALUATIONS

Safety evaluations will include monitoring of adverse events and serious adverse events, physical examinations, vital sign measurements, electrocardiograms, clinical laboratory tests, pregnancy testing, and checking of vital status.

VIROLOGY EVALUATIONS

SARS-CoV-2 positivity should be documented based on local testing on any specimen by RT-PCR or any other commercial or public health assay any time before randomization. This might require a local test using an NP swap obtained at screening.

In addition, NP swabs will be collected for assessment of viral load of SARS-CoV-2 virus. Furthermore, sequencing might be performed upon request of the virologist to determine mutations in the viral genome.

Blood and stool samples will also be collected to perform SARS-CoV-2 testing in a central laboratory and blood samples will be used to evaluate humoral immunity to SARS-CoV-2 by measuring SARS-CoV-2 specific antibodies (IgG, IgA, and IgM). Leftover samples may be used for exploratory virology research, to assess co-infections and/or exploratory biomarker research such as host RNA and protein testing.

PHARMACOKINETIC AND IMMUNOGENICITY EVALUATIONS

Serum samples will be used to evaluate the pharmacokinetics of infliximab, as well as antibodies to infliximab. Serum collected for pharmacokinetic and immunogenicity analyses may additionally be used to evaluate biomarkers, safety or efficacy aspects that address scientific questions relating to infliximab or SARS-CoV-2 infections.

GENETICS AND PHARMACOGENOMICS

An optional pharmacogenomic (host DNA) blood sample may be collected (preferably at baseline) from those participants who gave consent to allow for host pharmacogenomic research, where local regulations permit. Pharmacogenomic research may include expression quantitative trait locus (eQTL) mapping, single-nucleotide polymorphisms (SNPs) mapping and relevant whole genome sequencing related to the study intervention and/or SARS-CoV-2 infections. PHARMACODYNAMIC AND BIOMARKER EVALUATIONS

Blood samples will be collected to evaluate pharmacodynamic response and biomarkers that may be associated with safety, efficacy and PK of infliximab and/or disease caused by respiratory infections. Pharmacodynamic evaluations may include, but are not limited to, IL-6, TNFa, IL-1, pro- calcitonin, CRP, ferritin, LDH, and D-dimer concentrations. The study also includes collection of blood samples for exploratory analysis of host biomarkers including RNA profiling, proteomics, cellular profiling, including PBMC collection. Samples may be analyzed, under the supervision of the sponsor and results might be reported separately.

STATISTICAL METHODS

The primary analysis will be done when all participants reached Day 28 or discontinued earlier.

The final analysis will be done when all participants completed the study.

The efficacy endpoints will be analyzed on the Intent-to-Treat-infected (ITT-i) and by randomized treatment allocation. The ITT-i set consists of all participants who were randomized and treated and were confirmed to have SARS-CoV-2 infection.

All safety endpoints will be evaluated on the Safety Population, which consists of all participants who received at least one dose of study intervention and will be analyzed by treatment arm.

For all participants who receive study drug descriptive statistics will be provided. All demographic characteristics (e.g., age, race, ethnicity, height, body weight, body mass index) and other initial participant characteristics (e.g., physical examination, medical and surgical history, concomitant diseases) will be tabulated and analyzed descriptively. Subgroup analyses will be performed based on the stratification factors (age [<65 and >65 years of age] and use of invasive mechanical ventilation [yes/no]) and a selection of other major baseline parameters.

Sample Size Determination

The study will aim to enroll approximately 270 participants, with 180 participants in the infliximab treatment arm and 90 participants in the control arm.

For the sample size determination for the primary endpoint (time to improvement of at least 2 categories relative to Day 1 on the 6-point ordinal clinical recovery scale), it is assumed that the log transformed time to improvement (days) in survivors in the control arm follows a normal distribution with mean of log 14 and a standard deviation 0.47. Participants who die prior to Day 28 are treated as right censored at Day 28. The mortality in the control arm is assumed to be 50% by Day 28. Assuming a reduction in mortality from 50% to 35% (30% relative reduction) and a reduction of the median time to clinical improvement from 14 days to 10.5 days (25% reduction) in the surviving participants, at least 150 participants are required to have at least 80% power for a Gehan-Wilcoxon test, at a significance level of 5% two-sided.

The proportion of participants with a clinical improvement of at least 2 categories at Day 28 is a key secondary endpoint. In order to have 80% power to demonstrate also a significant difference between the treatment groups for this endpoint (46% vs 64% derived from the above assumptions), the study sample size will be 270. This will increase the power for the primary endpoint to 96%.

Analysis of the Primary Endpoint

The primary efficacy analysis will be based on the ITT-i analysis set and the primary efficacy endpoint is the ‘time to improvement of at least 2 categories relative to Day 1 on the 6- point ordinal clinical recovery scale (up to Day 28)’. Day 1 is defined as the worst category up to start of infusion and for the 12 hours subsequent to infusion on the day of study intervention administration. The improvement should be sustained until Day 28 (or discharge/discontinuation). Time to clinical improvement will be assessed during the 28-day period after study drug administration, with failure to reach clinical improvement or death before Day 28 considered as right-censored at Day 28.

This primary parameter will be analyzed by a stratified Gehan-Wilcoxon test (using the stratification factors). Kaplan-Meier curves, overall and by stratum will be used to graphically present the primary parameter. The sensitivity analyses will be defined in the SAP, but amongst others a stratified log-rank test will be applied.

Analyses of the Secondary Endpoints

First, the primary endpoint will be tested for superiority of infliximab over placebo at the 2-sided 5% significance level. If superiority is shown on the primary endpoint, then the key secondary endpoint will also be tested at the same significance level. All other statistical tests will be done as exploratory.

The proportion of participants with an improvement on Day 28 of at least 2 categories on the 6-point ordinal clinical recovery scale relative to Baseline is a key secondary endpoint. This parameter will be analyzed using a logistic regression model including the stratification factors.

All secondary endpoints will be analyzed graphically and descriptively as described in the statistical analysis plan. For continuous variables, descriptive statistics (n, mean, SD, median, minimum, maximum, and 95% confidence intervals [CIs]) will be calculated. For categorical variables, frequency tables and corresponding 95% CIs will be presented.

Safety analyses

All safety analyses will be made on the Safety Population.

All reported AEs will be included in the analysis. For each AE, the percentage of participants who experience at least 1 occurrence of the given event will be summarized by intervention group. Summaries, listings, datasets, or participant narratives may be provided, as appropriate, for those participants who die, who discontinue intervention due to an AE, or who experience a grade 3 or 4 AE or a serious AE.

The proportion of participants with SAEs, the proportion of participants with grade 3 or 4 AEs, the proportion of participants with severe or life-threatening, bacterial, invasive fungal, viral or opportunistic infections (other than SARS-CoV-2), the incidence of grade 3 and 4 neutropenia and lymphocytopenia, and the incidence of increased ALT >3xULN combined with increased bilirubin >2xULN will be analyzed using a logistic regression model.

Descriptive statistics will be calculated for clinical laboratory parameters, ECG parameters, vital signs, and physical examination findings.

Statistical testing might be applied to the secondary endpoints.

Interim Analysis

To enable an early safety assessment, recruitment will be halted after enrollment of the first 60 participants. As soon as the first 30 participants have been dosed and have had at least 7 days of follow-up, the Data Review Committee (DRC) will perform an interim data review. All data available at the time of the interim analysis will be included. Recruitment will be resumed upon a positive assessment of results of these interim data by DRC. Specific safety stopping rules will be specified in the DRC charter.

While futility or stopping of the study will be based on totality of emerging safety data, the DRC will consider either of the following non-binding futility criteria as recommendation for stopping the study.

Excess mortality in the treatment arm, beyond the realm of chance (Fisher’ s Exact test at a

1 -sided significance level of 10%).

Excess in “Worsening with 1 category” in the treatment arm, beyond the realm of chance

(Fisher’s exact test at a 1-sided significance level of 10%).

The DRC is also instructed to use their assessment of individual cases to evaluate even if data do not meet the criteria.

Additional interim analyses may be performed depending on the enrolment rate to guide further development and to support interactions with Health Authorities.

Other Analyses

Serum infliximab concentrations will be summarized using descriptive statistics. The concentrations below the lowest quantifiable sample concentration of the assay will be treated as zero in the summary statistics. All concentrations below the lowest quantifiable sample concentration of the assay or missing data will be labeled as such in the concentration data listing or statistical analysis dataset.

The incidence of antibodies to infliximab will be summarized for all participants in the ITTi population with appropriate samples for detection of antibodies to infliximab (i.e., participants with at least 1 sample obtained after their dose of infliximab).

Statistical approaches to explore correlations between clinical outcome and blood biomarkers vary and depend on the different data types of the applied technology platforms, as well as on the extent of observed differences between participants.

Analysis of the relationship between various blood biomarkers, such as cytokines, RNA or cellular profiling, and viral parameters, immunogenicity, pharmacokinetics, safety and clinical outcome may be conducted. Descriptive statistics for actual values and (relative) changes from baseline for the different blood biomarkers assessed, will be tabulated for each biomarker.

SARS-CoV-2 viral load in NP swabs, endotracheal, blood and stool samples will be measured by a qRT-PCR assay. These data will be analyzed graphically and descriptively as described in the statistical analysis plan.

DNA samples can be used for research related to infliximab or SARS-CoV-2 infection. Pharmacogenomic research may consist of the analysis of one or more candidate genes, of the analysis of genetic markers throughout the genome, or the analysis of the entire genome (as appropriate) to evaluate potential genetic associations with prognosis of clinical outcomes in patients and prediction of responsiveness to active treatment.

Screening

SARS-CoV-2 PCR positivity at any time at or before screening will be accepted. Results from local laboratory and X-ray assessments taken up to 2 days prior to screening will be accepted for screening assessments. Informed consent and screening (and SARS-CoV-2 PCR test, if SARS-CoV-2 test has not been done before) should occur within 24 hours prior to randomization. If all eligibility criteria are met at screening then randomization may occur on that same day. Participants need to receive study intervention preferably within 4 hours but no later than 6 hours after randomization. If the participant is on invasive mechanical ventilation, duration may not be >24 hours at time of screening.

Schedule of Activities

The Schedule of Activities (SoA; table below) summarizes the frequency and timing of respiratory function assessments, virology assessments, pharmacology assessments, exploratory biomarkers/pharmacogenomics, and safety measurements applicable to this study. All screening assessments should take place prior to randomization and all baseline assessments prior to study intervention administration. Results from the blood test, pregnancy test, and pulmonary X-ray, completed as standard of care, taken up to 2 days prior to screening will be accepted for screening assessments. SARS-CoV-2 positivity, as determined by real time-PCR or any other commercial or public health assay, in any specimen at any time prior to randomization is acceptable and this should not be repeated. All screening/baseline assessments should take place prior to randomization and/or study intervention administration. If multiple assessments are scheduled for the same timepoint, it is recommended that procedures be performed in the following sequence: ECG, oxygen saturation, vital signs, blood sampling. Blood collections for PK assessments should be kept as close to the specified time as possible. Other measurements may be done earlier than specified timepoints if needed. Actual dates and times of assessments will be recorded in the source documentation and CRF. The amount of blood drawn from each participant in this study is approximately 275 mL. Repeat or unscheduled samples may be taken for safety reasons or for technical issues with the samples.

Note: If multiple assessments are scheduled for the same timepoint, it is recommended that procedures be performed in the following sequence: ECG, oxygen saturation, vital signs, blood sampling. Baseline tests must be completed prior to study intervention administration.

Abbreviations: AE: adverse event; AESI: adverse event of special interest; DBP: diastolic blood pressure; ECG: electrocardiogram; Fi02: percentage of inspired oxygen; ICF: informed consent form; ICU: intensive care unit; PaCh: partial pressures of oxygen in arterial blood; PaCCh: partial pressure of carbon dioxide in arterial blood; PCR: polymerase chain reaction; SAE: serious adverse event; SaCh: arterial oxygen saturation; SBP: systolic blood pressure; SOC: standard of care; SpCh: peripheral capillary oxygen saturation Footnotes on Screening a. All screening and baseline assessments may take place on the same day. In such case, the tests do not need to be repeated. All screening assessments should take place prior to randomization and all baseline assessments prior to study intervention administration.

5 b. Results from the blood chemistry, hematology, and coagulation test, pregnancy test, and pulmonary X-ray or CT-scan, completed as SOC, taken up to 2 days prior to screening will be accepted as screening assessments. c. Participants need to receive study intervention preferably within 4 hours but no later than 6 hours after randomization. lOd. The post Day 28 assessment schedule will remain the same for participants still hospitalized after Day 28. e. Consent to be obtained before the first study-related activity. An exception is in the case of an emergency enrollment in which the informed consent can be obtained as soon as possible. f. If a participant's clinical status changes after screening but before the study intervention is given

15 such that he or she no longer meets all eligibility criteria, then the participant should be excluded from participation in the study. g. Please note that one of the inclusion criteria is “Requiring supplemental oxygen delivered at a flow >6 L/min, to sustain a Sp02 >93% regardless of device/route used (corresponds to category 3 or 4 on the 6-point ordinal scale)”. For these participant’s, confirm, if safe to do so, that the participant

20 has an SpCh < 93% on 4 L/min of supplemental oxygen, which constitutes a requirement or a need for at least 6 L/min. h. Medical history should include collecting onset of COVID-19 symptoms, prior therapy, and date of SARS-CoV-2 diagnosis if available. i. A pregnancy assessment is to be performed in women of childbearing potential only.

25j. Targeted physical examination includes lung auscultation and any examination as indicated by the participant’s medical history. Height and body weight are only to be measured at screening if not already available in the participant’s chart and if practically feasible. k. Supplemental oxygen/percentage of inspired oxygen (FiCh) use (if any) will be measured (simultaneously with SpCh, and also simultaneously with any time of blood gas measurements) to

30 monitor the participant’s status regarding gas exchange as applicable. The following will be recorded: Oxygen delivery device (e.g., nasal cannula, simple face mask, nonrebreather mask, high flow nasal cannula, non-invasive ventilation, invasive mechanical ventilation, extracorporeal life support, etc).

Oxygen flow rate in liters/min. - Record FiCh and Sp02 data 4 times per day, and at any time of blood gas measurements.

Record values that are sustained for at least 1 hour. For the analyses, the worst values sustained for at least 1 hour, on the highest level of oxygen supplementation method of the day, will be used.

If a participant is using more than one device (e.g., extracorporeal life support and invasive ventilation), information (the worst recording) from both devices will be recorded.

If a participant does not need oxygen supplement, this should also be recorded.

1. The final worst score of the Glasgow Coma Scale of the day needs to be recorded in the eCRF. The level of sedation of the participant will be derived from the type of medication entered for indication sedation on the Concomitant Medication page of the eCRF. m. Laboratory testing will be performed, this may include:

Hematology and liver parameters should be reported to the sponsor via eCRF data entry within approximately 72 hours from results becoming available. n. Culture results (bacterial, fungal, or viral) including site of infection, specimen source (bronchoalveolar lavage [BAL], tracheal aspirate, sputum, blood, urine, etc.) and timing of specimen collection, performed as part of participant’s work-up for new infections should be reported. Analyses will be performed by the local laboratory. o. For participants discharged prior to Day 28 a phone call will be conducted on Day 28 and during the post Day 28 Phase to assess the vital status, the occurrence of AEs, and the history of readmission since last contact. p. Whenever possible, vital status will be recorded if the participant is alive. If the participant is deceased, date and cause of mortality should be recorded in the eCRF. Death should be documented as an SAE. q. NP swabs will be used to collect secretions from participants to assess viral load of SARS-CoV- 2. For each participant, NP sampling should be done at approximately the same time (±4 hours) on each sampling day and from the same nostril. r. If an NP sample for detection of SARS-CoV-2 (local SOC) will be collected on the same day as the NP sample for quantification of SARS-CoV-2 (central lab), only one NP sample should be collected. The sample should be aliquoted and the remaining aliquots of the NP samples should be stored and sent to the central lab for quantification of SARS-CoV-2.

5s. SARS-CoV-2 positivity should be documented based on local testing on any specimen by RT- PCR or any other commercial or public health assay any time before randomization. This might require a local test using an NP swab obtained at screening. t. After randomization, SARS-CoV-2 positivity will be confirmed in a central lab by quantitative RT-PCR. The baseline sample needs to be collected predose, as close as possible to dosing. lOu. If the participant is intubated, endotracheal samples need to be taken at the same time as the NP swab. If taking both NP and endotracheal samples is not feasible, the NP sample should be given priority. v. Lab testing for detection of SARS-CoV-2 on the NP swab at day of hospital discharge. If SARS-CoV-2 positive, an additional NP swab will be taken every 7 days, if feasible for the site

15 and tested, until SARS-CoV-2 negative. w. Optional samples should be taken, unless for technical or clinical reason the additional blood draw is considered not possible. x. Includes plasma and serum samples for exploratory biomarker endpoint evaluation (including but not limited to TNFa, IL-6, IL-1, pro-calcitonin, CRP, ferritin, LDH and D-dimer serum

20 concentrations). y. Includes serum samples for measurement of PK of infliximab and antibodies to infliximab. On Day 1, a predose and a postdose (within 30 minutes after the end of infusion) sample should be collected. The postdose sample should be collected from the arm contralateral to that used for IV infusion. On Day 1 (predose) and Day 28 presence of antibodies to infliximab will be evaluated.

25 On Day 1 (postdose), Day 14, and Day 21 the PK of infliximab will be evaluated. z. For participants discharged after Day 28 an additional sample should be taken on the day of discharge. aa. PAXgene RNA tubes should always be used last for any blood draw. bb. An optional pharmacogenetics blood sample (DNA) will be obtained from those participants who

30 gave consent (where local regulations permit). cc. Sample can be also taken at any other time point after administration of study intervention on Day 1 dd. If viral RNA is detected in NP samples at day of discharge, all possible efforts will be made to follow-up participants and collect samples until viral RNA is negative, considering the current pandemic and related logistical challenges.

Inclusion Criteria

Each potential participant must satisfy all of the following criteria to be enrolled in the study:

1. Male or female >18 and <85 years of age.

2. Hospitalized

3. Has laboratory-confirmed SARS-CoV-2 infection as determined by real time-PCR or any other commercial or public health assay, at any time before randomization.

4. Evidence of infiltrates by chest X-ray, chest CT or chest auscultation (rales, crackles) Severe or critical COVTD-19 disease, defined as:

Severe disease: Requires supplemental oxygen administration by nasal cannula, simple face mask, or other similar oxygen delivery device (ie, above pre COVID baseline oxygen requirement, if any, by the participant).

Critical disease: Requires supplemental oxygen delivered by nonrebreather mask or high- flow nasal cannula OR use of non-invasive or invasive ventilation OR requiring treatment in an ICU.

AND at least one of the following:

• Requiring supplemental oxygen to sustain a Sp02 >93% regardless of device/route used (corresponds to category 3 or 4 on the 6 point ordinal scale).

OR

• Pa02/Fi02 ratio <300 mmHg while on invasive mechanical ventilation (with invasive mechanical ventilation for less than 24 hours prior to screening) (corresponds to category 5 on the 6-point ordinal scale).

• Must have a C-reactive protein level of >20 mg/L as measured at screening.

• Must understand English language and informed consent must be obtained (or their legally acceptable representative must understand English and sign based on local regulations) indicating that he or she understands the purpose of, and procedures required for, the study and is willing to participate in the study.

Exclusion Criteria

Any potential participant who meets any of the following criteria will be excluded from participating in the study:

1. On invasive mechanical ventilation for >24 hours at time of screening.

2. Meets local or global criteria to not receive mechanical ventilation or has designated themselves as DNR per a living will.

3. Received an investigational intervention (including investigational vaccines) or used an invasive investigational medical device within 30 days before the planned first dose of study intervention.

Note: the investigator must ensure that the participant is not enrolled in another COVID- 19 study with an investigational agent (apart from the exception specified below) prior to completion of Day 28 of the current study. Exception: participation in a single arm study or compassionate use study is allowed if it is conducted with one of the antiviral drug with demonstrated in vitro-ejfect against SARS-CoV-2, as mentioned in the CDC guidelines.

4. Suspected pulmonary embolus or Class 3 or 4 congestive heart failure as defined by the New York Heart Association Functional Classification (Section 10.8, Appendix 8: The New York Heart Association Classification System)

AND/OR

Evidence of active cardiac ischemia or history of myocardial infarction, unstable angina or acute coronary syndrome within 6 months prior to randomization.

5. Currently active and clinically significant uncontrolled arrhythmia.

Exception: Participants with premature ventricular contraction, supraventricular tachycardia or new atrial arrhythmias considered to be associated with the underlying disease may be included.

6. Uncontrolled hypertension (SBP above 180/110 mmHg).

7. Liver function impairment defined as Child Pugh Class B/C based on medical history.

8. Has congenital bleeding diathesis based on medical history.

9. Has a history of severe chronic obstructive pulmonary disease (COPD) or other severe chronic condition (i.e., asthma, cystic fibrosis, fibrotic lung disease) for which the degree of severity is defined as steroid dependent or that requires home oxygen supplementation, supportive non-invasive ventilation, is status/post lung volume reduction surgery (LVRS), or has known forced expiratory volume (FEV)l <50%

10. On renal replacement therapy (defined as peritoneal dialysis or hemodialysis)

11. Screening laboratory test result as follows:

• Absolute neutrophil count (ANC) <2.0 x 103 cells/pL (SI: <2.0 x 109 cells/L)

• Platelet count <100 x 103 cells/pL (SI: <50 x 109 cells/L)

• Estimated glomerular filtration rate (eGFR) <30 mL/min/1.73 m2

• Bilirubin >2xULN unless bilirubin rise is due to Gilbert’s syndrome or of non-hepatic origin

• ALT or AST >5xULN

12. Prothrombin time (PT)/international normalized ratio (INR) >1 5xULN and activated partial thromboplastin time (aPTT) >1.5xULN (unless abnormalities are unrelated to coagulopathy or bleeding disorder). Pregnant or breastfeeding, unless in the opinion of the investigator, the benefit outweighs the risks

13. Has active hepatitis B or C infection or HIV/AIDS based on medical history and/or concomitant medication.

14. Known active TB, history of incompletely treated TB, suspected or known extrapulmonary TB based on medical history and/or concomitant medication.

15. Evidence of active bacterial (including but not limited to bacterial pneumonia), fungal, viral or opportunistic infection other than SARS-CoV-2.

16. Known allergies, hypersensitivity, or intolerance to infliximab or its excipients or to other monoclonal antibodies or to any murine proteins.

17. Unlikely to be able to complete the study (including but not limited to: likely to be transferred to another hospital, surgery is anticipated to be necessary, in the opinion of the investigator unlikely to survive for >48 hours from screening)

18. Any condition for which, in the opinion of the investigator, participation would not be in the best interest of the participant (e.g., compromise the well-being) or that could prevent, limit, or confound the protocol-specified assessments.

19. Taken any disallowed therapies as noted in Section 6.8, Concomitant Therapy before the planned first dose of study intervention. 20. History of malignancy within 5 years before screening (exceptions are squamous and basal cell carcinomas of the skin and carcinoma in situ of the cervix, or malignancy, which is considered cured with minimal risk of recurrence).

21. Organ transplant recipient on immunosuppressant therapy

22. Have received tocilizumab or siltuximab within 15 weeks (105 days), sarilumab within 7 weeks (49 days), ustekinumab IV or guselkumab within 14 weeks (98 days), ustekinumab SC within 33 weeks (231 days), or an oral Janus Kinase inhibitor within 3 days of screening.

Drug Half-life 5 half-lives Source tocilizumab IV 21.5 days 107.5 days siltuximab IV 20.6 days 103 days sarilumab IV 200

10 days 50 days mg sarilumab IV 150

8 days 40 days mg U.S. FDA prescribing ustekinumab IV 19 days 95 days information guselkumab IV 15-18 days 75-90 days ustekinumab SC 14.9-45.6 days 74.5-228 days upadacitinib 8-14 hours 40-70 hours baricitinib 12 hours 60 hours tofacitinib XR 6-8 hours 30-40 hours

NOTE: Investigators should ensure that all study enrollment criteria have been met at screening. Retesting of abnormal laboratory values that may lead to exclusion will be allowed once. If a participant's clinical status changes after screening but before the study intervention is given such that he or she no longer meets all eligibility criteria, then the participant should be excluded from participation in the study. The required source documentation to support meeting the enrollment criteria are noted in Section 10.3, Appendix 3 Regulatory, Ethical, and Study Oversight Considerations.

Prestudy and Concomitant Therapy

Infliximab has immunomodulatory effects that may predispose participants to opportunistic infections. Therefore, that all participants are to be managed according to the latest version of CDC Information for Clinicians on Therapeutic Options for Patients with COVTD-19. Open-label or off-label use of agents that are intended to inhibit SARS-Cov-2 viral activity are permitted in the study, but they must be listed in the current CDC guidelines on Therapeutic Options for patients with COVTD-19.¹²

Use of an antiviral for treatment of COVTD-19 patients, should one become approved during the course of this clinical study, is permitted.

Disallowed concomitant medications

Conventional synthetic disease-modifying anti-rheumatic drugs/immunosuppressive agents: o Oral anti-rejection or immunomodulatory drugs (including tocilizumab) are disallowed from 6 months prior to randomization until the end of the study. o Treatment with other anti-TNFa, anti-IL-6, anti-IL6R antagonists, Janus kinase inhibitors, ustekinumab (anti IL-12/23), or anti IL-23 agents (guselkumab) is disallowed within 5 half-lives (Table 3) or from 30 days prior to randomization until the end of the study (whichever is longer).

Table 3: Treatment Half-life and 5 Half-lives

Drug Half-life 5 half-lives tocilizumab IV 21.5 days 107.5 days siltuximab IV 20.6 days 103 days sarilumab IV 200 mg 10 days 50 days sarilumab IV 150 mg 8 days 40 days ustekinumab IV 19 days 95 days guselkumab IV 15-18 days 75-90 days ustekinumab SC 14.9-45.6 days 74.5-228 days upadacitinib 8-14 hours 40-70 hours baricitinib 12 hours 60 hours tofacitinib XR 6-8 hours 30-40 hours

Source: Respective US Prescribing Information o Systemic treatment with disease-modifying anti-rheumatic drugs or immunosuppressive agents including methotrexate, bucillamine, azathioprine, oral cyclosporine A, tacrolimus, mycophenolate mofetil, leflunomide, oral or parenteral gold, and IL-lra (anakinra) is disallowed from 2 weeks prior to randomization until the end of the study.

Exceptions: sulfasalazine, hydroxychloroquine and chloroquine o The use of cyclophosphamide is disallowed from 12 weeks prior to randomization until the end of the study o Corticosteroids:

^■ potential participants on chronic (for >3 months in duration) prednisone in a dose higher than 10 mg/day or other oral corticosteroids at an equivalent dose for a non-COPD-related condition are not eligible for the study

^■ potential participants using oral corticosteroids for a non-COVTD-19-related condition are not eligible for the study. o The use of leflunomide is disallowed from 8 weeks prior to randomization until the end of the study. Potential participants who have undergone standard cholestyramine washout may qualify if it is done at least 4 weeks before randomization: cholestyramine at a dosage of 8 mg/3 times a day for at least 24 hours, or activated charcoal at a dosage of 50 mg/4 times a day for at least 24 hours.

• The participants should not have received an investigational intervention (including investigational vaccines) or used an invasive investigational medical device within 30 days of the planned dose of study intervention and should not receive any investigational medication, other than infliximab, prior to completion of Day 28.

Note: the investigator must ensure that the participant is not enrolled in another COVID-19 study with an investigational intervention (apart from the exception specified below) prior to completion of Day 28 of the current study.

Exception participation in a single arm study or compassionate use study is allowed if it is conducted with one of the agents with demonstrated in vitro-effect against SARS-CoV-2, as mentioned in the CDC guidelines (ref)

The sponsor must be notified in advance (or as soon as possible thereafter) of any instances in which prohibited therapies are administered.

Sample Collection and Handling

The actual dates and times of sample collection must be recorded in the CRF or laboratory requisition form. Refer to the SoA for the timing and frequency of all sample collections. Instructions for the collection, handling, storage, and shipment of samples are found in the laboratory manual that will be provided. Collection, handling, storage, and shipment of samples must be under the specified, and where applicable, controlled temperature conditions as indicated in the laboratory manual.

Efficacy Assessments

Efficacy assessments will be done per the SoA and will include 6-point ordinal Clinical Recovery Scale, level of consciousness (ACVPU), virology assessment, supplemental oxygen use, resting Sp02, arterial blood gas results, and pulmonary X-ray. Details for selected assessments are provided below.

Six-point Ordinal Clinical Recovery Scale

The 6-point ordinal clinical recovery scale provides 6 mutually exclusive conditions ordered from best to worst, and the score reflects the participant’s worst situation on the day of assessment. The hospital recovery scale categories are defined below. For ease of categorization, the categories are defined from worst to best.

Hospital Recovery Scale (and Definitions)

1. Not hospitalized

Any of the following:

- Discharged from the hospital the day of assessment

- Hospitalized at the day of assessment but ready for discharge on the day of assessment, as judged by the investigator (e.g., if still hospitalized in case of lack of bed availability in a skilled nursing facility, lack of social support at home).

2. Hospitalization, not requiring supplemental oxygen

3. Hospitalized, requiring supplemental oxygen

- Hospitalized on the day of assessment (including readmittance), and supplemental oxygen is required by the participant

Requiring supplemental oxygen is defined by:

- Receiving supplemental oxygen through a face mask or nasal cannula and not being able to sustain a blood oxygen saturation of >94% when breathing room air fori 5 minutes at any time on the day of assessment.

- Not receiving supplemental oxygen and having a blood oxygen saturation of <94% when breathing room air for 15 minutes at any measurement on the day of assessment.

4. Hospitalized, on non-invasive ventilation or high flow oxygen devices

5. Hospitalized, on invasive mechanical ventilation or ECMO

- Any oxygen support requiring intubation or extracorporeal oxygenation

- Invasive mechanical ventilation is used at any time on the day of assessment.

6. Death

- Participant died at any time on the day of assessment or earlier (all-cause mortality).

In addition, the site will be asked whether or not they are working under (a) restricted material resources for supplemental oxygenation/ventilation and/or (b) changes in hospital discharge policies.

Finally, a second 6-point ordinal clinical recovery scale is to be filled-in, with the assumption of working under unrestricted resources and conditions.

The frequency and timing of the assessment can be found in the SoA.

Level of Consciousness

The subject’s level of consciousness will be assessed using the ACVPU (Alert, Confusion, Voice, Pain, Unresponsive) scale. The patient’s mental status will be assessed and categorized into one of the following categories: alert, confusion, voice, pain, unresponsive.

• Alert: The subject is fully awake. This subject will have spontaneously opening of the eyes, will respond to voice, and will have motor function.

• New confusion: The subject may be alert but confused or disorientated. It is not always possible to determine whether the confusion is ‘new’ when a subject presents acutely ill. Such a presentation should always be considered ‘new’ until confirmed otherwise.

• Voice: The subject makes some kind of response when you talk to them, which could be in any of the 3 component measures of eyes, voice, or motor. The response could be as little as a grunt, moan, or slight move of a limb.

• Pain: The subject makes a response on any of the 3 component measures on the application of pain stimulus, such as a central pain stimulus (sternal rub) or a peripheral stimulus (squeezing the fingers).

• Unresponsive: Sometimes seen noted as 'Unconscious'; this outcome is recorded if the subject does not give any eye, voice or motor response to voice or pain.

The level of sedation should be recorded at the same time as the assessment of the level of consciousness. The frequency and timing of the assessment can be found in the SoA.

Virology Assessments

Collection of an NP sample at screening should be performed for detection of SARS- CoV-2, by a local laboratory. SARS-CoV-2 positivity, as determined by real time-PCR or any other commercial or public health assay, in any specimen at any time prior to randomization is acceptable and this should not be repeated.

In addition, nasopharyngeal swabs will be collected for central testing. A NP swab will be used to collect secretions from patients to explore quantification of viral load of SARS-CoV- 2 virus. At baseline, the presence of other respiratory pathogens, using multiplex PCR, will also be tested. Furthermore, sequencing might be performed upon request of the virologist to determine mutations in the viral genome.

For participant who are intubated, endotracheal samples need to be taken at the same time as the NP swabs. If taking both samples is not feasible, the NP should be given priority. Collected samples need to be sent to the central lab to explore quantification of viral load. Sequencing might be performed upon request of the virologist to determine mutations in the viral genome.

Only one sample should be collected if the NP sample for detection and the NP sample for quantification are collected on the same day. This sample should be aliquoted and the remaining aliquots of the NP samples should be stored and sent to the central lab for quantification of SARS-CoV-2. Leftover NP swabs and endotracheal samples may be used for exploratory biomarker analyses.

The frequency and timing of the assessments can be found in the SoA. Details about sample collection, processing, and shipping will be provided in the laboratory manual.

Plasma samples

In addition to NP swabs, plasma samples will be collected to assess SARS-CoV-2 viremia. Leftover plasma samples may be used for exploratory biomarker analyses. The frequency and timing of the assessments can be found in the SoA. Details about sample collection, processing, and shipping will be provided in the laboratory manual. Supplemental Oxygen Use

Supplemental oxygen/percentage of inspired oxygen (F1O2) use (if any) will be measured to monitor the patient’s status regarding gas exchange as applicable. The following will be recorded:

• Oxygen delivery device (e.g., nasal cannula, simple face mask, non-rebreather mask, high flow nasal cannula, non-invasive ventilation, invasive mechanical ventilation, extracorporeal life support, etc)

• Oxygen flow rate in liters/min

• Fi02 (if receiving high flow nasal cannula, non-invasive ventilation, invasive mechanical ventilation, or extracorporeal life support)

• If a patient is using more than one device (e.g., extracorporeal life support and invasive ventilation), information (the worst recording) from both devices will be recorded separately

• If a patient does not need oxygen supplement, this should also be recorded The frequency and timing of the assessment can be found in the SoA.

Resting Sp0₂

Resting Sp0₂ will be measured to assess arterial oxyhemoglobin saturation. SpO will be measured using a fingertip or similar non-invasive device, while patient is stable, following 5 minutes of rest (inactivity) in supine, semi-recumbent, or sitting position and will only be measured in the presence of a good SpO wave form. SpO must be measured simultaneously with recorded supplemental oxygen/FiC^ data. SpO must be measured simultaneously with recorded supplemental oxygen/FiC^ data. For participants receiving invasive or non-invasive mechanical ventilation, peripheral oxygen saturation should be measured with the ventilatory support in place, and it should be recorded. The frequency and timing of the assessment can be found in the SoA.

Arterial Blood Gas Test

An arterial blood test will be conducted to assess the following parameters:

• pH: acid-base balance of blood

• Pa02: partial pressure of oxygen in arterial blood

• PaC02: partial pressure of carbon dioxide in arterial blood • Sa02: arterial oxygen saturation

Results should be recorded in arterial blood gas results electronic clinical report form (eCRF). The frequency and timing of the assessment can be found in the SoA.

Safety Assessments

Safety and tolerability will be evaluated throughout the study from obtaining confirmed consent onwards until the last study-related activity. Adverse events will be reported and followed by the investigator. Any clinically significant abnormalities persisting at the end of the study/early withdrawal will be followed by the investigator until resolution or until a clinically stable condition is reached. The study will include the following evaluations of safety and tolerability according to the time points provided in the SoA.

Physical Examinations

A targeted physical examination will be performed as indicated in the SoA. A targeted physical examination includes lung auscultation and any examination as indicated by the patient’s medical history. Height and body weight are only to be measured at screening if not already available in the participant’s chart and if practically feasible. Clinically significant findings should be documented as AE in the eCRF if they are serious, possibly related to the study drug or correspond to an adverse event of interest.

Vital Signs

Temperature, pulse rate, respiratory rate, and blood pressure (SBP/DBP) will be assessed. The frequency and timing for each assessment can be found in the SoA. Body temperature will be measured according to local hospital protocols and according to the manufacturer’s instructions for use of the device. Body temperature should be measured using the same method each time: temperature should be measured after at least 5 minutes of rest (supine or sitting) and before taking antipyretics or more than 4 hours after the last dose of antipyretics. Blood pressure and pulse/heart rate measurements will be assessed with a completely automated device. Manual techniques will be used only if an automated device is not available. Confirmatory vital signs measurements can be performed if inconsistent with a prior measurement.

Any clinically relevant abnormalities or changes in severity should be documented as AE in the eCRF if they are serious, possibly related to the study drug or correspond to an adverse event of interest. Electrocardiograms

Participants should rest in a supine position for at least 5 minutes before ECG collection and should refrain from talking or moving arms or legs. If multiple assessments are scheduled for the same time point as ECG recording, the procedures should preferably be performed in the following order: ECG(s), oxygen saturation, vital signs, blood draw. Heart rate will be recorded from the ventricular rate and the PR, QRS, and QT (identify QT interval corrected for heart rate [QTc] using Bazett’s formula [QTcB] or QTc using Fridericia’s formula [QTcF]) intervals will be recorded in the eCRF. The ECG strips or reports will be retained with the source. The frequency and timing for each assessment can be found in the SoA. Any clinically relevant abnormalities or changes in severity should be documented as AE in the eCRF if they are serious, possibly related to the study drug or correspond to an adverse event of interest.

Clinical Safety Laboratory Assessments

Blood samples for serum chemistry and hematology will be collected. The investigator must review the laboratory results, document this review, and record any clinically relevant changes occurring during the study in the AE section of the CRF. The laboratory reports must be filed with the source documents. Any clinically relevant abnormalities or changes in severity should be documented as AE in the eCRF if they are serious, possibly related to the study drug or correspond to an adverse event of special interest (AESI).

Pregnancy Testing

At screening, absence of pregnancy in women of childbearing potential should preferably be confirmed by a negative highly sensitive serum (b-human chorionic gonadotropin [PhCG]) test. The result of a prior serum pregnancy test that occurred within 2 calendar days (as part of SOC) before obtaining consent can be used in lieu of the screening requirement. If the timeframe does not allow for results of a serum test (in emergency situations), a urine rapid pregnancy test is acceptable.

Vital Status

During the post Day 28 follow-up phone call visits vital status of study participants will be recorded. If the participant is deceased, date and cause of mortality should be recorded. Death should be documented as SAE in the eCRF.

Adverse Events, Serious Adverse Events, and Other Safety Reporting

Timely, accurate, and complete reporting and analysis of safety information, including AEs, SAEs, and product quality complaints (PQCs), from clinical studies are crucial for the protection of participants, investigators, and the sponsor, and are mandated by regulatory agencies worldwide. The sponsor has established Standard Operating Procedures in conformity with regulatory requirements worldwide to ensure appropriate reporting of safety information; all clinical studies conducted by the sponsor or its affiliates will be conducted in accordance with those procedures. Adverse events will be reported by the participant (or, when appropriate, by a caregiver, surrogate, or the participant's legally acceptable representative) for the duration of the study.

Time Period and Frequency for Collecting Adverse Event and Serious Adverse Event Information All Adverse Events

All adverse events and special reporting situations, whether serious or non-serious, will be reported from the time a signed and dated ICF is obtained until completion of the participant's last study-related procedure, which may include phone call contact for follow-up of safety. SAEs, including those spontaneously reported to the investigator within 30 days after the last dose of study intervention, must be reported using the SAE Form. The sponsor will evaluate any safety information that is spontaneously reported by an investigator beyond the time frame specified in the protocol.

Serious Adverse Events

All SAEs, as well as PQCs, occurring during the study must be reported to the appropriate sponsor contact person by study-site personnel within 24 hours of their knowledge of the event. Serious adverse events, including those spontaneously reported to the investigator within 30 days after the EOT (Week 16) phone call visit, must be reported using a Serious Adverse Event form. The sponsor will evaluate any safety information that is spontaneously reported by an investigator beyond the time frame specified in the protocol.

Information regarding SAEs will be transmitted to the sponsor using the Serious Adverse Event Form and Safety Report Form of the CRF, which must be completed and reviewed by a physician from the study site, and transmitted to the sponsor within 24 hours. The initial and follow-up reports of an SAE should be transmitted electronically or by facsimile (fax). Telephone reporting should be the exception and the reporter should be asked to complete the appropriate form(s) first. Method of Detecting Adverse Events and Serious Adverse Events

Care will be taken not to introduce bias when detecting AEs or SAEs. Open-ended and nonleading verbal questioning of the participant is the preferred method to inquire about adverse event occurrence. During the hospital stay, in case participants are non-responsive, investigators will report AEs as specified.

Solicited Adverse Events

Solicited adverse events are predefined local (at the injection site) and systemic events for which the participant is specifically questioned.

Unsolicited Adverse Events

Unsolicited AEs are all AEs for which the participant is not specifically questioned. Follow-up of Adverse Events and Serious Adverse Events

The investigator is obligated to perform or arrange for the conduct of supplemental measurements and evaluations as medically indicated to elucidate the nature and causality of the AE, SAE, or PQC as fully as possible. This may include additional laboratory tests or investigations, histopathological examinations, or consultation with other health care professionals.

Regulatory Reporting Requirements for Serious Adverse Events

The sponsor assumes responsibility for appropriate reporting of AEs to the regulatory authorities. The sponsor will also report to the investigator (and the head of the investigational institute where required) all suspected unexpected serious adverse reactions (SUSARs). The investigator (or sponsor where required) must report SUSARs to the appropriate Independent Ethics Committee/Institutional Review Board (IEC/IRB) that approved the protocol unless otherwise required and documented by the IEC/IRB. A SUSAR will be reported to regulatory authorities unblinded. Participating investigators and IEC/IRB will receive a blinded SUSAR summary, unless otherwise specified.

Pregnancy

All initial reports of pregnancy in female participants or partners of male participants must be reported to the sponsor by the study-site personnel within 24 hours of their knowledge of the event using the appropriate pregnancy notification form. Abnormal pregnancy outcomes (e.g., spontaneous abortion, fetal death, stillbirth, congenital anomalies, ectopic pregnancy) are considered serious adverse events and must be reported using a serious adverse event reporting form. Follow-up information regarding the outcome of the pregnancy and any postnatal sequelae in the infant will be required.

Adverse Events of Special Interest

Adverse events of special interest for the single IV administration of infliximab (5 mg/kg) are provided below.

Pharmacokinetics and Immunogenicity

Serum samples will be used to evaluate the pharmacokinetics of infliximab, as well as antibodies to infliximab. Serum collected for pharmacokinetic and immunogenicity analyses may additionally be used to evaluate biomarkers, safety or efficacy aspects that address scientific questions relating to infliximab or SARS-CoV-2 infections. Genetic analyses will not be performed on these serum samples. Details about sample collection, processing, and shipping will be provided in the laboratory manual.

Evaluations At visits where PK and/or immunogenicity will be evaluated, one venous blood draw will be collected as specified in the Schedule of Activities (SoA). Analytical Procedures Pharmacokinetics

Serum samples will be analyzed to determine concentrations of infliximab using a validated, specific, and sensitive immunoassay method by or under the supervision of the sponsor.

Immunogenicity

The detection and characterization of antibodies to infliximab will be performed using a validated immunoassay method by or under the supervision of the sponsor.

Pharmacokinetic Parameters and Evaluations

If feasible, a population PK approach will be used to characterize the disposition characteristics of infliximab. Total systemic clearance (CL) and volume of distribution (V) after IV administration may be estimated from population PK modeling using nonlinear mixed effects model (NONMEM) approach.

Immunogenicity Assessments

Antibodies to infliximab will be evaluated in serum samples collected on Day 1 predose, Day 28, and additionally on the day of discharge if participants are hospitalized longer than 28 days. Serum samples will be screened for antibodies binding to infliximab and the titer of confirmed positive samples will be reported. Other analyses may be performed to verify the stability of antibodies to infliximab and/or further characterize the immunogenicity of infliximab. Genetics and Pharmacogenomics

An optional pharmacogenomic (host DNA) blood sample may be collected (preferably at baseline) from those participants who gave consent to allow for host pharmacogenomic research, where local regulations permit. Pharmacogenomic research may include expression quantitative trait locus (eQTL) mapping, single-nucleotide polymorphisms (SNPs) mapping and whole genome sequencing that is related to relevant genes, study intervention and/or SARS-CoV-2 infections.

Participant participation in pharmacogenomic research is optional.

Pharmacodynamics and Biomarkers

The frequency and timing of the assessment can be found in the Schedule of Activities

(SoA). Sample collection for exploratory endpoint evaluation

Blood samples will be collected to evaluate pharmacodynamic response and biomarkers that may be associated with safety, efficacy and PK of infliximab and/or SARS-CoV-2 infection. Pharmacodynamic evaluations may include, but are not limited to, IL-6, TNFa, IL-1, pro calcitonin, CRP, ferritin, LDH, and D-dimer concentrations.

The study includes collection of blood samples for exploratory analysis of host biomarkers including RNA profiling, proteomics, cellular profiling, including PBMC collection. Samples may be analyzed, under the supervision of the sponsor and results might be reported separately. Samples can only be used for research related to infliximab or SARS-CoV-2 infection and/or to develop tests/assays related to infliximab or SARS-CoV-2 infection. This may include target pathway of TNFa inhibition, and the impact on pneumonia and respiratory illness associated with SARS-CoV-2 infection. Analysis of exploratory biomarkers may be conducted at the sponsor’s discretion and results may be reported separately from this study. STATISTICAL CONSIDERATIONS

Statistical analysis will be done by the sponsor or under the authority of the sponsor. A general description of the statistical methods to be used to analyze the efficacy and safety data is outlined below. Specific details will be provided in the Statistical Analysis Plan (SAP).

Statistical Hypotheses

The primary hypothesis of this study is that infliximab in combination with SOC results in a statistically significant shorter time to improvement (defined as an improvement of at least 2 categories relative to Baseline on the 6-point ordinal clinical recovery scale) versus placebo in combination with SOC, in participants with confirmed severe or critical COVTD-19 disease.

Sample Size Determination

The study will aim to enroll approximately 270 participants, with 180 participants in the infliximab treatment arm and 90 participants in the control arm. For the sample size determination for the primary endpoint (time to improvement of at least 2 categories relative to Baseline on the 6-point ordinal clinical recovery scale), it is assumed that the log transformed time to improvement (days) in survivors in the control arm follows a normal distribution with mean of log 14 and a standard deviation 0.47. Participants who die prior to Day 28 are treated as right censored at Day 28. The mortality in the control arm is assumed to be 50% by Day 28. Assuming a reduction in mortality from 50% to 35% (30% relative reduction) and a reduction of the median time to clinical improvement from 14 days to 10.5 days (25% reduction) in the surviving participants, at least 150 participants are required to have at least 80% power for a Gehan-Wilcoxon test, at a significance level of 5% two-sided. The proportion of participants with a clinical improvement of at least 2 categories at Day 28 is a key secondary endpoint. In order to have 80% power to demonstrate also a significant difference between the treatment groups for this endpoint (46% vs 64% derived from the above assumptions), the study sample size will be 270. This will increase the power for the primary endpoint to 96%.

Populations for Analysis Sets

For purposes of analysis, the following populations are defined:

The efficacy endpoints will be analyzed on the Intent-to-Treat-infected (ITT-i) and by randomized treatment allocation. The ITT-i set consists of all participants who were randomized and treated and were confirmed to have SARS-CoV-2 infection. All safety endpoints will be evaluated on the Safety Population, which consists of all participants who received study intervention and will be analyzed by treatment arm. Pharmacokinetic data will be evaluated on participants in the ITT-i set who received infliximab.

Statistical Analyses

The statistical analysis plan will be finalized prior to database lock of the primary analysis and it will include a more technical and detailed description of the statistical analyses described in this section. This section is a summary of the planned statistical analyses of the most important endpoints including primary and key secondary endpoints. The primary analysis will be done when all participants reached Day 28 or discontinued earlier. The final analysis will be done when all participants completed the study.

A DRC will be established for this study. The DRC will review interim data as soon as the first 30 participants have been dosed and have had at least 7 days of follow-up. Additional interim analyses might be performed depending on the enrollment rate to guide further development and to support interactions with Health Authorities. A pooled analysis may be considered as an exploratory analysis if the placebo group in this study is comparable with that in the sponsor’s sirukumab study (CNTO136COV2001).

General Considerations

For all participants who receive study intervention descriptive statistics will be provided. All demographic characteristics (e.g., age, race, ethnicity, height, body weight, body mass index) and other initial participant characteristics (e.g., physical examination, medical and surgical history, concomitant diseases) will be tabulated and analyzed descriptively. Subgroup analyses will be performed based on the stratification factors (age [<65 and >65 years of age] and use of invasive mechanical ventilation [yes/no]) and a selection of other major baseline parameters.

A DRC will be established as noted in Committees Structure in Section 10.3, Appendix 3 Regulatory, Ethical, and Study Oversight Considerations.

Primary Endpoint

The primary efficacy analysis will be based on the ITT-i analysis set and the primary efficacy endpoint is the ‘time to improvement of at least 2 categories relative to Baseline on the 6-point ordinal clinical recovery scale. The improvement should be sustained until Day 28 (or discharge/discontinuation). Time to clinical improvement will be assessed during the 28-day period after study intervention administration, with failure to reach clinical improvement or death before Day 28 considered as right-censored at Day 28. This primary parameter will be analyzed by a stratified Gehan-Wilcoxon test (using the stratification factors). Kaplan-Meier curves, overall and by stratum will be used to graphically present the primary parameter. The sensitivity analyses will be defined in the SAP, but amongst others a stratified log-rank test will be applied. First, the primary endpoint will be tested for superiority of infliximab over placebo at the 2-sided 5% significance level. If superiority is shown on the primary endpoint, then the key secondary endpoint will also be tested at the same significance level. All other statistical tests will be done as exploratory.

Secondary Endpoints

All secondary endpoints will be analyzed graphically and descriptively as described in the statistical analysis plan. For continuous variables, descriptive statistics (n, mean, SD, median, minimum, maximum, and 95% confidence intervals [CIs]) will be calculated. For categorical variables, frequency tables and corresponding 95% CIs will be presented.

Key Secondary Endpoint

The proportion of participants with an improvement on Day 28 of at least 2 categories on the 6-point ordinal clinical recovery scale relative to Baseline is a key secondary endpoint. This parameter will be analyzed using a logistic regression model including the stratification factors. Other Secondary Endpoints

To compare the proportion of participants with an improvement on Day 28 of at least 1 category on the 6-point ordinal clinical recovery scale relative to Baseline, proportion of participants with a worse score on the 6-point ordinal clinical recovery scale relative to Baseline, mortality rates, other ordinal endpoints, and proportion of participants on ECMO, a logistic regression model will be used. Stratification factors will be added to the model.

In addition, the following statistical testing will be done: Total length of hospitalization, total time on invasive mechanical ventilation, number of ventilation free days, and total time on ECMO will be analyzed by the stratified Wilcoxon Rank-Sum test and using the stratification factors. Corresponding 95% CIs will be derived using the Hodges-Lehmann approach.

Time to improvement of at least 2 categories and other ‘time to event’ parameters will be analyzed using the stratified Gehan-Wilcoxon test.

A proportional odds model will be used to analyze the 6-point ordinal clinical recovery scale of the clinical status on each day up to Day 28. Changes in the SOC after treatment administration will be tabulated by treatment group. Other secondary parameters might be added in the SAP.

Exploratory Endpoints

Time to viral negativity will be analyzed analogously to that of the primary efficacy parameter. SARS-CoV-2 viral load in NP swabs, endotracheal, blood and stool samples will be measured by a qRT-PCR assay. These data will be analyzed graphically and descriptively as described in the statistical analysis plan.

Other exploratory parameters might be added in the SAP.

Safety Analyses

All safety analyses will be made on the Safety Population.

Adverse Events

The verbatim terms used in the eCRF by investigators to identify adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA). Any AE occurring at or after the initial administration of study intervention is considered to be treatment- emergent. All reported AEs will be included in the analysis. For each AE, the percentage of participants who experience at least 1 occurrence of the given event will be summarized by intervention group. Summaries, listings, datasets, or participant narratives may be provided, as appropriate, for those participants who die, who discontinue intervention due to an AE, or who experience a grade 3 or 4 AE or a serious AE. The proportion of participants with SAEs, the proportion of participants with grade 3 or 4 AEs, the proportion of participants with severe or life-threatening, bacterial, invasive fungal, viral or opportunistic infections (other than SARS-CoV-2), the incidence of grade 3 and 4 neutropenia and lymphocytopenia, and the incidence of increased ALT >3xULN combined with increased bilirubin >2xULN will be analyzed using a logistic regression model.

Clinical Laboratory Tests

Laboratory data will be summarized by type of laboratory test. The laboratory abnormalities will be determined per the criteria specified in the DMID Adult Toxicity Table (Section 10.7, Appendix 7) and in accordance with the normal ranges of the clinical laboratory if no gradings are available. Descriptive statistics will be calculated for all laboratory analyte at baseline and for observed values and changes from baseline at each scheduled time point. A listing of participants with any laboratory results outside the reference ranges will be provided. A listing of participants with any markedly abnormal laboratory results will also be provided.

Safety Criteria for Acute Hepatic Impairment:

• ALT or AST >8xULN

• ALT or AST >5xULN for more than 2 weeks

• ALT or AST >3xULN and (TBL >2xULN or INR >1.5)

• ALT or AST >3xULN with the appearance of fatigue, nausea, vomiting, right upper quadrant pain or tenderness, fever, rash, and/or eosinophilia (>5%)

Participants with a history of hepatitis B and C viral infection and baseline negative PCR that develop elevations in liver function tests (should be evaluated for viral reactivation).

Electrocardiogram

Electrocardiogram data will be summarized by ECG parameter. Descriptive statistics will be calculated at baseline and for observed values and changes from baseline at each scheduled time point. Frequency tabulations of the abnormalities will be made.

Vital Signs

Vital signs including temperature, pulse/heart rate, respiratory rate, and blood pressure (systolic and diastolic) will be summarized over time, using descriptive statistics and/or graphically. The percentage of participants with values beyond clinically important limits will be summarized. Physical Examinations

Descriptive statistics of changes from baseline will be summarized at each scheduled time point. Physical examination findings will be summarized at each scheduled time point. Descriptive statistics will be calculated at baseline and for observed values and changes from baseline at each scheduled time point. Frequency tabulations of the abnormalities will be made.

Other Analyses Pharmacokinetic Analyses

Serum infliximab concentrations will be summarized using descriptive statistics. The concentrations below the lowest quantifiable sample concentration of the assay will be treated as zero in the summary statistics. All concentrations below the lowest quantifiable sample concentration of the assay or missing data will be labeled as such in the concentration data listing or statistical analysis dataset. If feasible, population PK analysis of serum concentration-time data of infliximab may be performed using nonlinear mixed-effects modeling. Data may be combined with other selected studies to support a relevant structural model. Available baseline participant characteristics (e.g., demographics, laboratory variables, etc.) may be tested as potential co variates affecting PK parameters. The results of the population PK analysis will be presented in a separate report.

Pharmacodynamic and Biomarkers Analyses

Analysis of the relationship between various blood biomarkers, such as cytokines, RNA or cellular profiling, and viral parameters, immunogenicity, pharmacokinetics, safety and clinical outcome may be conducted. Descriptive statistics for actual values and (relative) changes from baseline for the different blood biomarkers assessed, will be tabulated for each biomarker at each applicable time point specified (see Schedule of Activities (SoA)). Statistics include n, mean, SD, CV, geometric mean, median, minimum, and maximum. Statistical approaches to explore correlations between clinical outcome and blood biomarkers vary and depend on the different data types of the applied technology platforms, as well as on the extent of observed differences between participants. Analyses will be conducted at the sponsor’s discretion and may be reported separately from this study. Immunogenicity Analyses

The incidence of antibodies to infliximab will be summarized for all participants in the ITTi population with appropriate samples for detection of antibodies to infliximab (ie, participants with at least 1 predose and the Day 28 sample obtained after their dose of infliximab).

Virology Analyses

SARS-CoV-2 viral load summaries will be presented with descriptive statistics by intervention arm. If the virology endpoint is continuous, the descriptive statistics will include the number of participants, mean, standard deviation (SD), median, and range. If the virology endpoint is binary or categorical, the frequency distribution with the number and percentage of participants in each category will be calculated. For time-to-event variables, a summary table including number of participants included in the analysis, number of participants censored, 25th and 75th percentiles and median time-to event will be shown by intervention arm. Graphic displays will also be used to summarize the data. Comparison between viral load in NP, endotracheal, blood and stool samples might be performed.

Amino acid and/or nucleic acid substitutions in the SARS-CoV-2 genome will be tabulated and described.

Additional exploratory characterization may be performed and reported separately

Pharmacogenomic Analyses

DNA samples can be used for research related to infliximab or SARS-CoV-2 infection. Pharmacogenomic research may consist of the analysis of one or more candidate genes, of the analysis of genetic markers throughout the genome, or the analysis of the entire genome (as appropriate) to evaluate potential genetic associations with prognosis of clinical outcomes in patients and prediction of responsiveness to active treatment.

Results will be presented in a separate report.

Interim Analysis

To enable an early safety assessment, recruitment will be halted after enrollment of the first approximately 60 participants. As soon as the (estimated) first 30 participants have been dosed and have had at least 7 days of follow-up, the DRC will perform an interim data review. All data available at the time of the interim analysis will be included. Recruitment will be resumed upon a positive assessment of results of these interim data by DRC. Specific safety stopping rules will be specified in the DRC charter. While futility or stopping of the study will be based on totality of emerging safety data, the DRC will consider either of the following non-binding futility criteria as recommendation for stopping the study.

Excess mortality in the treatment arm, beyond the realm of chance (Fisher’ s Exact test at a 1 -sided significance level of 10%).

Excess in “Worsening with 1 category” in the treatment arm, beyond the realm of chance

(Fisher’s exact test at a 1-sided significance level of 10%).

The DRC is also instructed to use their assessment of individual cases to evaluate even if data do not meet the criteria. Additional interim analyses may be performed depending on the enrolment rate to guide further development and to support interactions with Health Authorities.

Definitions

The term “comprising” encompasses “including” as well as “consisting” e.g., a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.

The word “substantially” does not exclude “completely” e.g., a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

The term “about” in relation to a numerical value x means, for example, x±10%. In one embodiment, the word “about” may be omitted.

All references cited herein are incorporated by reference in their entirety.

Claims

Claims

1. A method for treating a human patient with a SARS-CoV-1 and/or SARS-CoV-2 infection comprising administering to the patient an effective amount of a pharmaceutical composition comprising an anti-TNFa agent.

2. The method of claim 1, wherein the anti-TNFa agent is an anti-TNFa antibody that blocks binding of TNFa to TNFa receptor.

3. The method of claim 1 or 2, wherein the patient is showing symptoms of COVTD-19 including severe hypoxemia and is at risk for developing Acute Respiratory Distress Syndrome (ARDS).

4. The method of claim 1 or 2, wherein the patient is showing symptoms of COVTD-19 with ARDS onset.

5. The method according to any of claims 2-4, wherein the antibody comprises infliximab or portion thereof that specifically binds TNFa.

6. The method according to any of claims 2-5, wherein the anti-TNFa antibody is administered to the patient according to a dosing regimen wherein the dosing regimen comprises at least one administration cycle and wherein for each of the at least one administration cycle, the anti-TNF antibody is administered to the patient in a dose between 1 and lOmg/kg of the patient’s body weight.

7. The method of any of claims 1-6, wherein the administration of the anti-TNFa antibody results in an improvement of at least one category relative to reference on a 6-point clinical recovery scale of clinical status selected from the group consisting of: not hospitalized, hospitalization with the patient not requiring supplemental oxygen, hospitalization with non- invasive ventilation or high flow oxygen device, hospitalization with invasive mechanical ventilation or extracorporeal membrane oxygenation (ECMO) and death.

8. The method of claim any of claims 1-7, wherein the the administration of the anti-TNFa antibody results in an improvement of the at least one category for at least 48 hours and up to 28 days.

9. The method of any of claims 1-8, wherein the antibody is administered to the patient in simultaneous or sequential combination with one or more antiviral agents.

10. The method of claim 9, wherein the one or more antiviral agents is independently selected from the group consisting of small molecules, vaccines, proteins, plasma derived agents.

11. The method of claim 10, wherein the one or more antiviral agents comprises a small molecule.

12. The method of claim 11, wherein the small molecule(s) is/are independently selected from the group consisting of favipiravir, remdesivir, ifenprodil, chloroquine, umifenovir, APN01, galidesivir, ritonavir, BPI 002, OYA1 and SNG001.

13. The method of claim 12, wherein the patient has previously been administered an antiviral agent but the antiviral agent has failed to treat or infection with SARS-CoV-1 and/or SARS- CoV-2.

14. An isolated anti-TNFa antibody for use according to any of claims 1-13, wherein the human has previously been administered an antiviral agent but the antiviral agent has failed to treat or infection with SARS-CoV-1 and/or SARS-CoV-2.

15. Products comprising an anti-TNFa antibody and an antiviral agent as listed in any one of claims 9-12 as a combined preparation for simultaneous, separate or sequential use in the treatment of infection of a human with SARS-CoV-1 and/or SARS-CoV-2.

16. A method for evaluating the efficacy of an anti-TNFa molecule wherein the schedule of activities recited below are followed:

17. A method for evaluating the safety and efficacy of a molecule for treating COVID-19 wherein the objectives and endpoints recited below are used to assess the efficacy of said molecule: