Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
  • G01N33/6857—Antibody fragments
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/52—Assays involving cytokines
  • G01N2333/525—Tumor necrosis factor [TNF]

Definitions

• Tumor necrosis factor alpha is a cytokine produced by numerous cell types, including monocytes and macrophages, that was originally identified based on its ability to induce the necrosis of certain mouse tumors. Subsequently, a factor termed cachectin, associated with cachexia, was shown to be identical to TNFa. TNFa has been implicated in the pathophysiology of a variety of other human diseases and disorders, including shock, sepsis, infections, autoimmune diseases, RA, Crohn’s disease, transplant rejection and graft-versus-host disease.
• hTNFa human TNFa
• therapeutic strategies have been designed to inhibit or counteract hTNFa activity.
• antibodies that bind to, and neutralize hTNFa have been sought as a means to inhibit hTNFa activity.
• Some of the earliest of such antibodies were mouse monoclonal antibodies (mAbs), secreted by hybridomas prepared from lymphocytes of mice immunized with hTNFa (see e.g., U.S. Pat. No. 5,231,024 to Moeller et al.).
• mouse anti- hTNFa antibodies often displayed high affinity for hTNFa and were able to neutralize hTNFa activity
• their use in vivo has been limited by problems associated with the administration of mouse antibodies to humans, such as a short serum half-life, an inability to trigger certain human effector functions, and elicitation of an unwanted immune response against the mouse antibody in a human (the“human anti-mouse antibody” (HAMA) reaction).
• HAMA human anti-mouse antibody
• Biological therapies have been applied to the treatment of autoimmune disorders such as rheumatoid arthritis.
• autoimmune disorders such as rheumatoid arthritis.
• four TNFa inhibitors REMICADETM
• CIMZIA ® certolizumab pegol
• CD moderate to severe Crohn’s disease
• TNFa inhibitors can induce an immune response to the drug and lead to the production of autoantibodies such as human anti-chimeric antibodies (HACA), human anti-humanized antibodies (HAHA), and human anti-mouse antibodies (HAMA).
• HACA, HAHA, or HAMA immune responses can be associated with hypersensitive reactions and dramatic changes in pharmacokinetics and biodistribution of the immunotherapeutic TNFa inhibitor that preclude further treatment with the drug.
• the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFa drug in a sample, the method comprising: contacting the sample with a TNFa labeled with a first fluorophore;
• the methods are useful for measuring the levels of
• REMICADETM infliximab
• INFLECTRA Infliximab-dyyb
• RENFLEXIS Infliximab- abda
• FLIXABI Infliximab Biosimilar
• REMSIMA Infliximab Biosimilar
• ENBREL TM etanercept
• HUMIRA TM adalimumab
• AMJEVITA Adalimumab-atto
• IMRALDI Adalimumab Biosimilar
• CYLTEZO Adalimumab Biosimilar
• HYRTMOZ Aldalimumab Biosimilar
• HULIO Adalimumab Biosimilar
• CIMZIA ® certolizumab pegol
• the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFa drug in a sample, the method comprising: contacting the sample with a TNFa with a first fluorophore;
• exciting the sample have dual labeled anti-TNFa drug using a light source to detect a fluorescence emission signal associated with fluorescence resonance energy transfer (FRET).
• FRET fluorescence resonance energy transfer
• the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFa drug in a sample, the method comprising: contacting the sample with a complex comprising an anti-TNFa drug labeled with a first fluorophore and an isolated TNFa labeled with a second fluorophore, wherein the complex emits a fluorescence emission signal associated with fluorescence resonance energy transfer (FRET) when excited using a light source;
• FRET fluorescence resonance energy transfer
• the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFa drug autoantibody (autoantibody) in a sample, the method comprising: contacting the sample with a first labeled anti-TNFa drug or Fab fragment with a donor fluorophore; contacting the sample with a second labeled anti-TNFa drug or Fab fragment with an acceptor fluorophore;
• FIG. 1 A illustrates one embodiment of the present disclosure for detecting anti- TNFa drug concentration.
• FIG. IB illustrates one embodiment of the present disclosure for detecting anti- TNFa drug concentration.
• FIG. 2 illustrates a standard curve generated using methods of the present disclosure.
• FIG. 3 illustrates one embodiment of a donor fluorophore of the present disclosure.
• FIG. 4 illustrates one embodiment of an acceptor fluorophore of the present disclosure.
• FIG. 5 illustrates donor and acceptor wavelengths in one embodiment of the present disclosure.
• Tb-H22TRENIAM-5LIO-NHS emission profile is shown (490 nm, 545 nm, 580 nm and 620 nm).
• Acceptor emission peaks are shown in (AF488, second arrow from left), (AF546, fourth arrow from left ) and (AF647, seventh arrow from the left i.e., first arrow on the right).
• FIG. 6 illustrates a trFRET adalimumab (ADA) assay which reaches equilibrium after 3 minutes. Eight concentrations ranging from 0 pg/mL to 50 pg/m were tested.
• Reagents were mixed with a sample and read at different time points as shown.
• FIG. 7A illustrates one embodiment of the present disclosure for detecting anti-drug antibody (autoantibodies).
• FIG. 7B illustrates one embodiment of the present disclosure for detecting anti-drug antibody (autoantibodies).
• FIG. 8 illustrates a standard curve generated using methods of the present disclosure.
• FIG. 9 illustrates a comparison of methods of the present disclosure
• FIG. 10 illustrates a standard curve generated using methods of the present disclosure.
• FIG. 11 illustrates a comparison of methods of the present disclosure and commercially available method.
• Activated acyl as used herein includes a -C(0)-LG group.“Leaving group” or “LG” is a group that is susceptible to displacement by a nucleophilic acyl substitution (z.e., a nucleophilic addition to the carbonyl of -C(0)-LG, followed by elimination of the leaving group).
• Representative leaving groups include halo, cyano, azido, carboxylic acid derivatives such as t-butylcarboxy, and carbonate derivatives such as i-Bu0C(0)0-.
• An activated acyl group may also be an activated ester as defined herein or a carboxylic acid activated by a carbodiimide to form an anhydride (preferentially cyclic) or mixed anhydride -OC(0)R a or - OC(NR a )NHR b (preferably cyclic), wherein R a and R b are members independently selected from the group consisting of C1-C 6 alkyl, C1-C 6 perfluoroalkyl, C1-C 6 alkoxy, cyclohexyl, 3- dimethylaminopropyl, or N-morpholinoethyl.
• Preferred activated acyl groups include activated esters.
• Activated ester includes a derivative of a carboxyl group that is more susceptible to displacement by nucleophilic addition and elimination than an ethyl ester group (e.g ., an NHS ester, a sulfo-NHS ester, a PAM ester, or a halophenyl ester).
• an ethyl ester group e.g ., an NHS ester, a sulfo-NHS ester, a PAM ester, or a halophenyl ester.
• Representative carbonyl substituents of activated esters include succinimidyloxy (- OC4H4NO2), sulfosuccinimidyloxy (-OC4H3NO2SO3H), -1-oxybenzotriazolyl (-OC6H4N3); 4- sulfo-2,3,5,6-tetrafluorophenyl; or an aryloxy group that is optionally substituted one or more times by electron-withdrawing substituents such as nitro, fluoro, chloro, cyano,
• Preferred activated esters include succinimidyloxy,
• FRET partners refer to a pair of fluorophores consisting of a donor fluorescent compound such as cryptate and an acceptor compound such as Alexa 647, when they are in proximity to one another and when they are excited at the excitation wavelength of the donor fluorescent compound, these compounds emit a FRET signal. It is known that, in order for two fluorescent compounds to be FRET partners, the emission spectrum of the donor fluorescent compound must at least partially overlap the excitation spectrum of the acceptor compound.
• the preferred FRET-partner pairs are those for which the value R0 (Forster distance, distance at which energy transfer is 50% efficient) is less than or equal to 100 A.
• FRET Fluorescence resonance energy transfer
• FRET Formster resonance energy transfer
• FRET signal refers to any measurable signal representative of FRET between a donor fluorescent compound and an acceptor compound.
• a FRET signal can therefore be a variation in the intensity or in the lifetime of luminescence of the donor fluorescent compound or of the acceptor compound when the latter is fluorescent.
• anti-TNFa drug or“TNFa inhibitor” as used herein is intended to encompass agents including proteins, antibodies, antibody fragments, fusion proteins (e.g., Ig fusion proteins or Fc fusion proteins), multivalent binding proteins (e.g., DVD Ig), small molecule TNFa antagonists and similar naturally or non-naturally-occurring molecules, and/or recombinant and/or engineered forms thereof, that, directly or indirectly, inhibit TNF a activity, such as by inhibiting interaction of TNFa with a cell surface receptor for TNFa, inhibiting TNFa protein production, inhibiting TNFa gene expression, inhibiting TNFa secretion from cells, inhibiting TNFa receptor signaling or any other means resulting in decreased TNFa activity in a subject.
• fusion proteins e.g., Ig fusion proteins or Fc fusion proteins
• multivalent binding proteins e.g., DVD Ig
• small molecule TNFa antagonists and similar naturally or non-naturally-occurring molecules e.g
• anti-TNFa drug or“TNFa inhibitor” preferably includes agents which interfere with TNFa activity.
• TNFa inhibitors include etanercept (ENBREL TM , Amgen), infliximab (REMICADE TM , Johnson and Johnson), human anti-TNF monoclonal antibody adalimumab (D2E7/HUMIRA TM , Abbott
• TNF a activity such that when administered to a subject suffering from or at risk of suffering from a disorder in which TNF a activity is detrimental (e.g., RA), the disorder is treated.
• a disorder in which TNF a activity is detrimental e.g., RA
• anti-drug antibody refers to an antibody of an anti-TNFa drug or a TNFa inhibitor.
• TNFa inhibitors such as etanercept (ENBRELTM, Amgen), infliximab (REMICADE TM , Johnson and Johnson), human anti-TNF monoclonal antibody adalimumab (D2E7/HUMIRA TM , Abbott Laboratories), certolizumab pegol (CIMZIA ® , UCB, Inc.), CDP 571 (Celltech), and CDP 870 (Celltech).
• an“anti-drug antibody” is an antibody of an antibody.
• Bio-Rad product codes for anti-infliximab antibodies include HCA214, HCA215, HCA216, and HCA216P.
• “TNFa” is an“antigen,” which is a molecule or a portion of the molecule capable of being bound by an anti-TNFa antibody.
• TNFa will react, in a highly selective manner, with an anti-TNFa antibody.
• TNFa is a sufficient length having an epitope of TNF a that is capable of binding anti-TNFa antibodies, fragments and regions thereof.
• the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFa drug in a sample, the method comprising: contacting the sample with a TNFa labeled with a first fluorophore;
• the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFa drug in a sample, the method comprising: contacting the sample with a TNFa with a first fluorophore;
• the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFa drug in a sample, the method comprising:
• a complex comprising an anti-TNFa drug labeled with a first fluorophore and an isolated TNFa labeled with a second fluorophore, wherein the complex emits a fluorescence emission signal associated with fluorescence resonance energy transfer (FRET) when excited using a light source;
• FRET fluorescence resonance energy transfer
• anti-TNFa drug in the sample competes with the anti-TNFa drug labeled with a first fluorophore.
• the greater the decrease in the fluorescence emission signal relative to the fluorescence emission signal initially emitted by the complex indicates a greater amount of anti-TNFa drug in the sample
• the methods are useful for measuring or monitoring the presence, level or concentration of biologies such as REMICADE TM (infliximab),
• INFLECTRA Infliximab-dyyb
• RENFLEXIS Infliximab-abda
• FLIXABI Infliximab Biosimilar
• REMSIMA Infliximab Biosimilar
• ENBREL TM etanercept
• HUMIRA TM adalimumab
• AMJEVITA Adalimumab-atto
• IMRALDI Alalimumab Biosimilar
• CYLTEZO Adalimumab Biosimilar
• HYRIMOZ Alalimumab Biosimilar
• HULIO Adalimumab Biosimilar
• CIMZIA ® certolizumab pegol
• the FRET assay is a time-resolved FRET assay.
• the fluorescence emission signal or measured FRET signal is directly correlated with the biological phenomenon studied.
• the level of energy transfer between the donor compound and the acceptor fluorescent compound is proportional to the reciprocal of the distance between these compounds to the 6 th power.
• the distance Ro (corresponding to a transfer efficiency of 50%) is in the order of 1, 5, 10, 20 or 30 nanometers.
• the sample is a biological sample.
• suitable biological samples include, but are not limited to, whole blood, plasma, serum, blood cells, cell samples, urine, spinal fluid, sweat, tear fluid, saliva, skin, mucous membrane, and hair.
• whole blood, plasma, serum, blood cells and such are preferred, and whole blood, blood cells, and such are particularly preferred.
• Whole blood includes samples of whole blood-derived blood cell fractions admixed with plasma. With regard to these samples, samples subjected to pretreatments such as hemolysis, separation, dilution, concentration, and purification can be used.
• the biological sample is a whole blood or a serum sample.
• the first fluorophore is a donor and the second fluorophore is an acceptor.
• the first fluorophore is an acceptor and the second fluorophore is a donor.
• the FRET energy donor compound is a cryptate, such as a lanthanide cryptate.
• the FRET assay format for the anti-TNFa drug is a FRET anti-antibody bridge assay.
• the assay includes contacting the sample with a TNFa labeled with a first fluorophore, such as a donor fluorophore.
• the sample is also contacted with an anti-drug antibody or Fab fragment labeled with a second fluorophore, such as an acceptor fluorophore.
• These contacting steps can be simultaneous or sequential.
• the sample incubates for a time sufficient to obtain a dual labeled anti-TNFa drug.
• the sample is then excited using a light source to detect a fluorescence emission signal associated with fluorescence resonance energy transfer (FRET).
• FRET fluorescence resonance energy transfer
• the FRET assay format for example, of adalimumab (e.g., DRUG, an anti-TNFa drug) is an Amplified Sandwich TR-FRET assay.
• the TNFa labeled with a donor fluorophore binds to the anti-TNFa drug (e.g. adalimumab).
• the anti-adalimumab Fab with an acceptor fluorophore preferably binds to the adalimumab Fab region when TNFa is also bound.
• the anti-adalimumab Fab with an acceptor fluorophore binds to the adalimumab Fab region only when TNFa is also bound.
• Each analyte (anti-TNFa drug, e.g., adalimumab) contains two Fab regions, enabling multiple bindings of TNFa donor and anti-adalimumab Fab acceptor, thus yielding twice the possible FRET signal per analyte.
• one or more TNFa labeled donors can bind and one or more anti -Fab acceptors can bind.
• each Fab region on the analyte can contain the TNFa donor and anti-adalimumab Fab acceptor in close proximity, enabling energy transfer producing an“amplified” or increased FRET signal.
• An increasing FRET signal indicates increased concentration of the anti-TNFa drug.
• the cryptate has an absorption wavelength between about 300 nm to about 400 nm.
• cryptate dyes have four fluorescence emission peaks at about 490 nm, about 545 nm, about 580 nm, and 620 nm.
• the cryptate is compatible with fluorescein-like (green zone) molecules, Cy5, DY-647- like (red zone) acceptors, Allophycocyanin (APC), or Phycoeruythrin (PE) to perform TR- FRET experiments.
• the introduction of a time delay between a flash excitation and the measurement of the fluorescence at the acceptor emission wavelength allows to discriminate long lived from short-lived fluorescence and to increase signal-to- noise ratio.
• the detection device detects time-resolved (tr) fluorescent signal from both the donor and FRET acceptor emission.
• Time-resolved (tr) FRET is a technique to improve signal to noise by removing short-lived fluorescent signals originating from the sample.
• the donor fluorophore is excited using a pulse of light.
• the emission from both the donor and acceptor signals are read after a time delay from the end of the excitation pulse. Noise is reduced as background fluorescence from nonspecific sources decay more rapidly than the emitted light from the donor allowing the acceptor signal to be read long after the nonspecific fluorescence has passed.
• the assay uses a donor fluorophore consisting of terbium bound within a cryptate.
• the terbium cryptate can be excited with a 365 nm UV LED.
• the terbium cryptate emits at four (4) wavelengths within the visible region.
• the assay uses the lowest donor emission energy peak of 620 nm as the donor signal within the assay.
• the acceptor fluorophore when in very close proximity, is excited by the highest energy terbium cryptate emission peak of 490 nm causing light emission at 520 nm. Both the 620 nm and 520 nm emission wavelengths are measured independently in a device or instrument and results can be reported as RFU ratio 620/520.
• the methods are useful for measuring the levels of
• autoantibodies including, but not limited to, human anti-chimeric antibodies (HACA), human anti-humanized antibodies (HAHA), and human anti-mouse antibodies (HAMA) in a sample, e.g., from a subject receiving anti-TNFa drug therapy.
• HACA human anti-chimeric antibodies
• HAHA human anti-humanized antibodies
• HAMA human anti-mouse antibodies
• the autoantibodies can be non neutralizing or neutralizing autoantibodies.
• the present disclosure provides an assay method for detecting the presence or amount of an anti-TNFa drug autoantibody (autoantibody) in a sample, the method comprising: contacting the sample with a first labeled anti-TNFa drug or Fab fragment with a donor fluorophore;
• the terbium cryptate molecule“Lumi4-Tb” from Lumiphore, marketed by Cisbio bioassays is used as the cryptate donor.
• An activated ester can react with a primary amine on an antibody to make a stable amide bond.
• a maleimide on the cryptate and a thiol on the antibody can react together and make a thioether.
• Alkyl halides react with amines and thiols to make alkylamines and thioethers, respectively. Any derivative providing a reactive moiety that can be conjugated to an antibody can be utilized herein.
• Microcycles are suitable for use in the present disclosure.
• This publication contains cryptate molecules useful for labeling biomolecules. As disclosed therein, certain of the cryptates have the structure as follows:
• a terbium cryptate useful in the present disclosure is shown below:
• the cryptates that are useful in the present invention are disclosed in WO 2018/130988, published July 19, 2018. As disclosed therein, the compounds of Formula I are useful as FRET donors in the present disclosure:
• R and R 1 are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, alkyl optionally substituted with one or more halogen atoms, carboxyl, alkoxycarbonyl, amido, sulfonato, alkoxycarbonylalkyl or alkylcarbonylalkoxy or alternatively, R and R 1 join to form an optionally substituted cyclopropyl group wherein the dotted bond is absent;
• R 2 and R 3 are each independently a member selected from the group consisting of hydrogen, halogen, SCbH, -SO2-X, wherein X is a halogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, or an activated group that can be linked to a biomolecule, wherein the activated group is a member selected from the group consisting of a halogen, an activated ester, an activated acyl, optionally substituted alkyl sulfonate ester, optionally substituted arylsulfonate ester, amino, formyl, glycidyl, halo, haloacetamidyl, haloalkyl, hydrazinyl, imido ester, isocyanato, isothiocyanato, maleimidyl, mercapto, alkynyl, hydroxyl
• R 4 are each independently a hydrogen, C1-C6 alkyl, or alternatively, 3 of the R 4 groups are absent and the resulting oxides are chelated to a lanthanide cation;
• Q '-Q 4 are each independently a member selected from the group of carbon or nitrogen.
• a FRET acceptor In order to detect a FRET signal, a FRET acceptor is required.
• the FRET acceptor has an excitation wavelength that overlaps with an emission wavelength of the FRET donor.
• the FRET signal of the acceptor is proportional to the concentration level of analyte present in the sample, such as a patient’s blood sample as interpolated from a known amount of calibrators i.e., a standard curve.
• acceptor molecules that can be used include, but are not limited to, fluorescein- like (green zone) acceptor, Cy5, DY-647, Alexa Fluor 488, Alexa Fluor 546,
• Donor and acceptor fluorophores having reactive moieties such as an NHS ester can be conjugated using a primary amine on an antibody.
• acceptors include, but are not limited to, cyanine derivatives, D2, CY5, fluorescein, coumarin, rhodamine, carbopyronine, oxazine and its analogs, Alexa Fluor fluorophores, Crystal violet, perylene bisimide fluorophores, squaraine fluorophores, boron dipyrromethene derivatives, NBD (nitrobenzoxadiazole) and its derivatives, DABCYL (4- ((4-(dimethylamino)phenyl)azo)benzoic acid).
• fluorescence can be characterized by wavelength, intensity, lifetime, polarization or a combination thereof.
• an activated ester (an NHS ester) of the donor or acceptor can react with a primary amine on an antibody to make a stable amide bond.
• a maleimide on the cryptate or the acceptor e.g., Alexa 647
• a thiol on the antibody can react together and make a thioether.
• Alkyl halides react with amines and thiols to make alkylamines and thioethers, respectively.
• Any derivative providing a reactive moiety that can be conjugated to an antibody can be utilized herein to make the first antibody labeled with a donor fluorophore specific for the analyte, as well as, the second antibody labeled with an acceptor fluorophore specific for analyte.
• the methods herein can use a variety of samples, which include a tissue sample, blood, biopsy, serum, plasma, saliva, urine, or stool sample.
• binding fragments or Fab fragments which mimic antibodies can also be prepared from genetic information by various procedures (see, e.g, Antibody Engineering: A Practical Approach, Borrebaeck, Ed., Oxford University Press, Oxford (1995); and Huse et al, J. Immunol., 149:3914-3920 (1992)).
• phage display technology to produce and screen libraries of polypeptides for binding to a selected target antigen (see, e.g, Cwirla et al, Proc. Natl. Acad. Sci. USA, 87:6378-6382 (1990); Devlin et al, Science, 249:404-406 (1990); Scott et al, Science, 249:386-388 (1990); and Ladner et al, U.S. Patent No. 5,571,698).
• a basic concept of phage display methods is the establishment of a physical association between a polypeptide encoded by the phage DNA and a target antigen.
• This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide.
• the establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides.
• Phage displaying a polypeptide with affinity to a target antigen bind to the target antigen and these phage are enriched by affinity screening to the target antigen.
• the identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods, a polypeptide identified as having a binding affinity for a desired target antigen can then be synthesized in bulk by conventional means (see, e.g, U.S. Patent No. 6,057,098).
• the antibodies that are generated by these methods can then be selected by first screening for affinity and specificity with the purified polypeptide antigen of interest and, if required, comparing the results to the affinity and specificity of the antibodies with other polypeptide antigens that are desired to be excluded from binding.
• the screening procedure can involve immobilization of the purified polypeptide antigens in separate wells of microtiter plates. The solution containing a potential antibody or group of antibodies is then placed into the respective microtiter wells and incubated for about 30 minutes to 2 hours.
• microtiter wells are then washed and a labeled secondary antibody (e.g ., an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 minutes and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immobilized polypeptide antigen is present.
• a labeled secondary antibody e.g ., an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies
• the antibodies so identified can then be further analyzed for affinity and specificity.
• the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ, e.g., certain antibody combinations may interfere with one another sterically, assay performance of an antibody may be a more important measure than absolute affinity and specificity of that antibody.
• Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a polypeptide of interest and an adjuvant. It may be useful to conjugate the polypeptide of interest to a protein carrier that is immunogenic in the species to be immunized, such as, e.g, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent.
• a protein carrier that is immunogenic in the species to be immunized, such as, e.g, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent.
• Animals are immunized against the polypeptide of interest or an immunogenic conjugate or derivative thereof by combining, e.g, 100 pg (for rabbits) or 5 pg (for mice) of the antigen or conjugate with 3 volumes of Freund’s complete adjuvant and injecting the solution intradermally at multiple sites.
• the animals are boosted with about 1/5 to 1/10 the original amount of polypeptide or conjugate in Freund’s incomplete adjuvant by subcutaneous injection at multiple sites.
• the animals are bled and the serum is assayed for antibody titer. Animals are typically boosted until the titer plateaus.
• the animal is boosted with the conjugate of the same polypeptide, but conjugation to a different immunogenic protein and/or through a different cross-linking reagent may be used.
• Conjugates can also be made in recombinant cell culture as fusion proteins.
• aggregating agents such as alum can be used to enhance the immune response.
• Monoclonal antibodies are generally obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
• the modifier“monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
• monoclonal antibodies can be made using the hybridoma method described by Kohler et al, Nature , 256:495 (1975) or by any recombinant DNA method known in the art (see, e.g., U.S. Patent No. 4,816,567).
• a mouse or other appropriate host animal e.g, hamster
• lymphocytes that produce or are capable of producing antibodies which specifically bind to the polypeptide of interest used for immunization.
• lymphocytes are immunized in vitro.
• the immunized lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (see, e.g., Coding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)).
• the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances which inhibit the growth or survival of the unfused, parental myeloma cells.
• a suitable culture medium that preferably contains one or more substances which inhibit the growth or survival of the unfused, parental myeloma cells.
• the culture medium for the hybridoma cells will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT -deficient cells.
• HGPRT medium hypoxanthine, aminopterin, and thymidine
• Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and/or are sensitive to a medium such as HAT medium.
• myeloma cell lines for the production of human monoclonal antibodies include, but are not limited to, murine myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center; San Diego, CA), SP-2 or X63-Ag8-653 cells
• the culture medium in which hybridoma cells are growing can be assayed for the production of monoclonal antibodies directed against the polypeptide of interest.
• the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as a radioimmunoassay (RIA) or an enzyme-linked immunoabsorbent assay (ELISA).
• RIA radioimmunoassay
• ELISA enzyme-linked immunoabsorbent assay
• the binding affinity of monoclonal antibodies can be determined using, e.g, the Scatchard analysis of Munson el al, Anal. Biochem., 107:220 (1980).
• the clones may be subcloned by limiting dilution procedures and grown by standard methods (see, e.g, Coding, Monoclonal Antibodies:
• Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
• the hybridoma cells may be grown in vivo as ascites tumors in an animal.
• the monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
• DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g, by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
• the hybridoma cells serve as a preferred source of such DNA.
• the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody, to induce the synthesis of monoclonal antibodies in the recombinant host cells. See, e.g., Skerra et al, Curr. Opin.
• the DNA can also be modified, for example, by substituting the coding sequence for human heavy chain and light chain constant domains in place of the homologous murine sequences (see, e.g, U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non
• monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in, for example, McCafferty et al., Nature, 348:552-554 (1990); Clackson et al., Nature, 352:624- 628 (1991); and Marks et al, J. Mol. Biol., 222:581-597 (1991).
• the production of high affinity (nM range) human monoclonal antibodies by chain shuffling is described in Marks et al, BioTechnology, 10:779-783 (1992).
• the use of combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries is described in
• human antibodies can be generated.
• transgenic animals e.g, mice
• transgenic animals e.g, mice
• JH antibody heavy-chain joining region
• phage display technology can be used to produce human antibodies and antibody fragments in vitro, using immunoglobulin variable (V) domain gene repertoires from unimmunized donors.
• V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as Ml 3 or fd, and displayed as functional antibody fragments on the surface of the phage particle.
• the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties.
• the phage mimics some of the properties of the B cell.
• Phage display can be performed in a variety of formats as described in, e.g., Johnson et al., Curr. Opin. Struct. Biol., 3:564-571 (1993).
• V- gene segments can be used for phage display. See, e.g, Clackson et al. , Nature , 352:624-628 (1991).
• a repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described in Marks et al, J. Mol. Biol., 222:581-597 (1991);
• human antibodies can be generated by in vitro activated B cells as described in, e.g, U.S. Patent Nos. 5,567,610 and 5,229,275.
• F(ab’)2 fragments can be isolated directly from recombinant host cell culture.
• the antibody of choice is a single chain Fv fragment (scFv). See, e.g, PCT Publication No. WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458.
• the antibody fragment may also be a linear antibody as described, e.g, in U.S. Patent No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.
• Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the same polypeptide of interest. Other bispecific antibodies may combine a binding site for the polypeptide of interest with binding site(s) for one or more additional antigens. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments ( e.g ., F(ab’)2 bispecific antibodies).
• antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
• the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy chain constant region (CHI) containing the site necessary for light chain binding present in at least one of the fusions.
• CHI first heavy chain constant region
• the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm.
• This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. See, e.g., PCT Publication No. WO 94/04690 and Suresh el a/. , Meth. Enzymol. , 121 :210 (1986).
• the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
• the preferred interface comprises at least a part of the CH3 domain of an antibody constant domain.
• one or more small amino acid side-chains from the interface of the first antibody molecule are replaced with larger side chains (e.g, tyrosine or tryptophan).
• Compensatory“cavities” of identical or similar size to the large side-chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side-chains with smaller ones (e.g, alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
• Bispecific antibodies include cross-linked or“heteroconjugate” antibodies.
• one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
• Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents and techniques are well-known in the art, and are disclosed in, e.g, U.S. Patent No. 4,676,980.
• bispecific antibodies can be prepared using chemical linkage.
• bispecific antibodies can be generated by a procedure in which intact antibodies are proteolytically cleaved to generate F(ab’)2 fragments (see, e.g, Brennan et al, Science , 229:81 (1985)). These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab’ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
• TAB thionitrobenzoate
• Fab’-TNB derivatives are then reconverted to the Fab’-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab’-TNB derivative to form the bispecific antibody.
• Fab’-SH fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies.
• a fully humanized bispecific antibody F(ab’)2 molecule can be produced by the methods described in Shalaby et al, ./. Exp. Med., 175: 217-225 (1992). Each Fab’ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.
• bispecific antibodies have been produced using leucine zippers. See, e.g., Kostelny et al. , J. Immunol., 148: 1547- 1553 (1992).
• the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab’ portions of two different antibodies by gene fusion.
• the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
• The“diabody” technology described by Hollinger et al. , Proc. Natl. Acad. Sci. USA,
• the fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites.
• VH heavy chain variable domain
• VL light chain variable domain
• Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers is described in Gruber et al, J.
• Antibodies with more than two valencies are also contemplated.
• trispecific antibodies can be prepared. See, e.g, Tutt et al, J. Immunol., 147:60 (1991).
• antibodies can be produced inside an isolated host cell, in the periplasmic space of a host cell, or directly secreted from a host cell into the medium. If the antibody is produced intracellularly, the particulate debris is first removed, for example, by centrifugation or ultrafiltration. Carter et al, BioTech., 10: 163-167 (1992) describes a procedure for isolating antibodies which are secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) for about 30 min. Cell debris can be removed by centrifugation.
• sodium acetate pH 3.5
• EDTA EDTA
• PMSF phenylmethylsulfonylfluoride
• supernatants from such expression systems are generally concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
• a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious
• the antibody composition prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography.
• the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
• Protein A can be used to purify antibodies that are based on human g ⁇ , g2, or g4 heavy chains (see, e.g., Lindmark el al. , ./. Immunol. Meth ., 62: 1-13 (1983)).
• Protein G is recommended for all mouse isotypes and for human g3 (see, e.g., Guss et al., EMBO J., 5: 1567-1575 (1986)).
• the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
• Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
• the antibody comprises a CH3 domain
• the Bakerbond ABXTM resin J. T. Baker; Phillipsburg, N.J. is useful for purification.
• the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction
• the methods are useful for detecting the amount of anti-TNFa drugs such as, e.g., antibodies including REMICADETM (infliximab), INFLECTRA
• anti-TNFa drugs such as, e.g., antibodies including REMICADETM (infliximab), INFLECTRA
• Infliximab-dyyb RENFLEXIS (Infliximab-abda), FLIXABI (Infliximab Biosimilar), REMSIMA (Infliximab Biosimilar), ENBREL TM (etanercept), HUMIRA TM (adalimumab), AMJEVITA (Adalimumab-atto), IMRALDI (Adalimumab Biosimilar), CYLTEZO (Adalimumab Biosimilar), HYRIMOZ (Adalimumab Biosimilar), HULIO (Adalimumab Biosimilar), and CIMZIA ® (certolizumab pegol).
• the phrase“high level of an anti-TNFa drug” includes drug levels of about 10 to about 100 ng/10 pL, about 10 to about 70 ng/10 pL, or about 10 to about 50 ng/10 pL. In other embodiments, the phrase“high level of anti-TNFa drug” includes drug levels greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ng/10 pL.
• the phrase“medium level of an anti-TNFa drug” includes drug levels of about 5.0 to about 50 ng/10 pL, about 5.0 to about 30 ng/10 pL, about 5.0 to about 20 ng/10 pL, or about 5.0 to about 10 ng/10 pL. In other embodiments, the phrase“medium level of anti-TNFa drug” includes drug levels of about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng/10 pL.
• the phrase“low level of an anti-TNFa drug” includes drug levels of about 0 to about 10 ng/10 pi, about 0 to about 8 ng/10 pL, or about 0 to about 5 ng/10 pi.
• the phrase“low level of an anti-TNFa drug” includes drug levels of about less than about 10, 9.0, 8.0, 7.0, 6.0, 5.0, 4.0, 3.0, 2.0, 1.0, or 0.5 ng/10 pi
• ADA includes the phrase“anti-drug antibody.” These or autoantibodies are an autoimmune reaction to the use of biologies. In other aspects, “ADA” is short hand for“Adalimumab.”
• the phrase“high level of an anti-drug antibody” includes anti drug antibody levels of about 3.0 to about 100 ng/10 pL, about 3.0 to about 50 ng/10 pL, about 10 to about 100 ng/10 pL, about 10 to about 50 ng/10 pL, or about 20 to about 50 ng/10 pL. In some other embodiments, the phrase“high level of anti-drug antibody” includes anti-drug antibody levels of about greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ng/10 pi.
• the phrase“medium level of an anti-drug antibody” includes anti drug antibody levels of about 0.5 to about 20 ng/10 pL, about 0.5 to about 10 ng/10 pi, about 2.0 to about 20 ng/10 pi, about 2.0 to about 10 ng/10 pL, about 2.0 to about 5.0 ng/10 pi, or about 2.0 to about 5.0 ng/10 pi.
• the phrase“low level of an anti-drug antibody” includes anti-drug antibody levels of about 0.0 to about 5.0 ng/10 pi, about 0.1 to about 5.0 ng/10 pL, about 0.0 to about 2.0 ng/10 pL, about 0.1 to about 2.0 ng/10 pL, or about 0.5 to about 2.0 ng/10 pi.
• the phrase“low level of anti-drug antibody” includes anti-drug antibody levels of about less than about 5.0, 4.0, 3.0, 2.0, 1.0, or 0.5 ng/10 m ⁇ .
• the present disclosure may further comprise recommending a course of therapy based upon the diagnosis, prognosis, or prediction.
• the present disclosure may further comprise administering to the individual a therapeutically effective amount of an anti-TNFa drug useful for treating one or more symptoms associated with disease and disorder in which TNFa has been implicated in the pathophysiology.
• the anti-TNFa drug can be administered alone or co-administered in combination with one or more additional anti-TNFa drugs and/or one or more drugs that reduce the side-effects associated with the anti-TNFa drug (e.g., an immunosuppressive agent).
• anti-TNFa drugs examples include, but are not limited to, REMICADETM (infliximab), INFLECTRA (Infliximab-dyyb), RENFLEXIS (Infliximab- abda), FLIXABI (Infliximab Biosimilar), REMSIMA (Infliximab Biosimilar), ENBREL TM (etanercept), HUMIRA TM (adalimumab), AMJEVITA (Adalimumab-atto), IMRALDI (Adalimumab Biosimilar), CYLTEZO (Adalimumab Biosimilar), HYRIMOZ (Adalimumab Biosimilar), HULIO (Adalimumab Biosimilar), CIMZIA ® (certolizumab pegol), and other biologic agents.
• the present disclosure advantageously enables a clinician to practice“personalized medicine” by guiding treatment decisions and informing therapy selection and optimization for anti-
• the anti-TNFa drug can be administered with a suitable pharmaceutical excipient as necessary and can be carried out via any of the accepted modes of administration.
• administration can be, for example, intravenous, topical, subcutaneous, transcutaneous, transdermal, intramuscular, oral, buccal, sublingual, gingival, palatal, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, or by inhalation.
• co-administer it is meant that an anti-TNFa drug is administered at the same time, just prior to, or just after the administration of a second drug (e.g., another anti-TNFa drug, a drug useful for reducing the side-effects of the anti- TNFa drug, etc.).
• a second drug e.g., another anti-TNFa drug, a drug useful for reducing the side-effects of the anti- TNFa drug, etc.
• a therapeutically effective amount of an anti-TNFa drug may be administered repeatedly, e.g., at least 2, 3, 4, 5, 6, 7, 8, or more times, or the dose may be administered by continuous infusion.
• the dose may take the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, pellets, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, foams, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.
• An individual can also be monitored at periodic time intervals to assess the concentrations or levels of various antibodies.
• the antibody levels at various time points, as well as the rate of change of the antibody levels over time is significant.
• the rate of increase of one or more antibodies (e.g., autoantibodies to anti-TNFa antibodies) in an individual over a threshold amount indicates the individual has a significantly higher risk of developing complications or risk of side-effects.
• Information obtained from serial testing in the form of a marker velocity i.e., the change in antibody levels over a time period
• the methods of the present disclosure also provide for identifying primary non- or low-responders, e.g, for treatment with a therapeutic monoclonal antibody.
• These primary non- or low-responders may, for example, be patients that happen to have an innate or a pre developed immunoglobulin response to the therapeutic agent.
• the therapeutic agent is a diagnostic antibody
• the identification of primary non- or -low responders can ensure the selection of a suitable therapeutic agent for each individual patient.
• a fluorescence spectrophotometer or fluorometer, fluorospectrometer, or fluorescence spectrometer measures the fluorescent light emitted from a sample at different wavelengths, after illumination with light source such as a xenon flash lamp. Fluorometers can have different channels for measuring differently-colored fluorescent signals (that differ in their wavelengths), such as green and blue, or ultraviolet and blue, channels.
• a suitable device includes an ability to perform a time-resolved fluorescence resonance energy transfer (TR-FRET) experiment.
• TR-FRET time-resolved fluorescence resonance energy transfer
• Suitable fluorometers can hold samples in different ways, including cuvettes, capillaries, Petri dishes, and microplates.
• the assays described herein can be performed on any of these types of instruments.
• the device has an optional microplate reader, allowing emission scans in up to 384-well plates, Others models suitable for use hold the sample in place using surface tension.
• Time-resolved fluorescence (TRF) measurement is similar to fluorescence intensity measurement.
• One difference, however, is the timing of the excitation / measurement process.
• the excitation and emission processes are simultaneous: the light emitted by the sample is measured while excitation is taking place.
• emission systems are very efficient at removing excitation light before it reaches the detector, the amount of excitation light compared to emission light is such that fluorescent intensity measurements exhibit elevated background signals.
• the present disclosure offers a solution to this issue.
• Time resolve FRET relies on the use of specific fluorescent molecules that have the property of emitting over long periods of time (measured in milliseconds) after excitation, when most standard fluorescent dyes (e.g.
• fluorescein emit within a few nanoseconds of being excited.
• a pulsed light source e.g., Xenon flash lamp or pulsed laser
• the FRET signal can be measured in different ways: measurement of the fluorescence emitted by the donor alone, by the acceptor alone or by the donor and the acceptor, or measurement of the variation in the polarization of the light emitted in the medium by the acceptor as a result of FRET.
• the FRET signal can be measured at a precise instant or at regular intervals, making it possible to study its change over time and thereby to investigate the kinetics of the biological process studied.
• the device disclosed in PCT/IB2019/051213, filed February 14, 2019 is used, which is hereby incorporated by reference. That disclosure in that application generally relates to analyzers that can be used in point-of-care (POC) settings to measure the absorbance and fluorescence of a sample with minimal or no user handling or interaction.
• POC point-of-care
• the disclosed analyzers provide advantageous features of more rapid and reliable analyses of samples having properties that can be detected with each of these two approaches. For example, it can be beneficial to quantify both the fluorescence and absorbance of a blood sample being subjected to a diagnostic assay.
• the hematocrit of a blood sample can be quantified with an absorbance assay, while the signal intensities measured in a FRET assay can provide information regarding other components of the blood sample.
• One apparatus disclosed in PCT/IB2019/051213 is useful for detecting an emission light from a sample, and absorbance of a transillumination light by the sample, which comprises a first light source configured to emit an excitation light having an excitation wavelength.
• the apparatus further comprises a second light source configured to emit an excitation light having an excitation wavelength.
• the apparatus further comprises a first light detector configured to detect the excitation light, and a second light detector configured to detect the emission light and the transillumination light.
• the apparatus further comprises a dichroic mirror configured to (1) epi-illuminate the sample by reflecting at least a portion of the excitation light, (2) transmit at least a portion of the excitation light to the first light detector, (3) transmit at least a portion of the emission light to the second light detector, and (4) transmit at least a portion of the transillumination light to the second light detector.
• One of the provided cuvettes comprises a hollow body enclosing an inner chamber having an open chamber top.
• the cuvette further comprises a lower lid having an inner wall, an outer wall, an open lid top, and an open lid bottom. At least a portion of the lower lid is configured to fit inside the inner chamber proximate to the open chamber top.
• the lower lid comprises one or more (e.g., two or more) containers connected to the inner wall, wherein each of the containers has an open container top. In certain aspects, the lower lid comprises two or more such containers.
• the lower lid further comprises a securing means connected to the hollow body.
• the cuvette further comprises an upper lid wherein at least a portion of the upper lid is configured to fit inside the lower lid proximate to the open lid top.
• Example 1 illustrates the measurement of detection of anti-TNFa drug biologies in blood and other matrices.
• FRET Fluorescence resonance energy transfer
• TRF time-resolved fluorometry
• TR-FRET Time-resolved FRET
• TNF-alpha molecule is labeled with a donor fluorophore and an anti-TNF drug Fab fragment labeled with an acceptor fluorophore
• TR-FRET can occur in the presence of anti-TNF-alpha drug (FIG. IB).
• each Fab region on the analyte can contain the TNF-a donor and Anti-adalimumab Fab acceptor in a close proximity, enabling greater energy transfer producing an Amplified FRET signal.
• the increase in FRET signal of the acceptor is proportional to the amount of antibody drug present in the patients’ blood or feces as interpolated from a known amount of calibrators.
• FIG. 2 illustrates a trFRET adalimumab (DRUG) calibration curve. Whole blood samples are serially diluted and used as calibrators.
• a donor fluorophore H22TRENIAM-5LIO-NHS (structure in FIG. 3) can be used to label TNF-alpha.
• the acceptor molecules that can be used include but are not limited to: AlexaFluor 488, AlexaFluor 546 and AlexaFluor 647.
• FIG. 4 shows the structure of
• Lumi4 has 4 spectrally distinct peaks, at about 490 nm, about 545 nm about 580, and about 620 nm (FIG. 5), which can be used for energy transfer.
• Donor and acceptor fluorophores are conjugated using primary amines on TNF-alpha and the Fab fragment of an anti-TNF-alpha drug antibody respectively.
• FIG. 6 illustrates a trFRET adalimumab (DRUG) assay reaches equilibrium after 3 minutes. Eight concentrations (0 pg/mL to 50 pg/ml) are tested. Reagents were mixed with a sample and read at different time points as shown.
• DRUG trFRET adalimumab
• Example 2 illustrates the measurement of detection of anti-TNFa drug
• autoantibodies may reduce the drug’s efficacy and/or induce adverse effects.
• autoantibodies have been found not only in patients treated with the chimeric antibody infliximab, but also in patients treated with the humanized antibody adalimumab. Monitoring of autoantibodies and drug levels in individual patients may help optimize treatment and dosing of the patient.
• FIG. 7A illustrates a method of the disclosure for detecting anti-drug antibody (autoantibodies).
• An anti-TNFa drug such as infliximab (IFX) can be labeled with a donor and another infliximab (IFX) can be labeled with an acceptor. Once bound to the autoantibodies human anti-chimeric antibodies (HACA) and the donor is excited, FRET occurs.
• HACA human anti-chimeric antibodies
• FIG. 7B the present disclosure provides methods for detecting anti-drug antibody (autoantibodies) using the Fab portion of anti-TNFa drugs (e.g. such as infliximab (IFX)).
• a Fab portion of an anti-TNFa drug such as infliximab (IFX) can be labeled with a donor and another Fab portion of infliximab (IFX) can be labeled with an acceptor.
• IFX infliximab
• HACA human anti-chimeric antibodies
• Example 3 illustrates the measurement of infliximab using the methods of this disclosure.
• a standard curve for infliximab was generated using the methods described herein.
• a TNF-alpha molecule was labeled with a donor fluorophore and an anti- TNF drug Fab fragment was labeled with an acceptor fluorophore.
• TR-FRET occurred in the presence of infliximab.
• the increase in FRET signal of the acceptor is proportional to the amount of drug present in the patients’ sample. The results are tabulated below and shown in FIG. 8.
• Example 4 illustrates a comparison of measuring infliximab using the methods of this disclosure versus Homogenous Mobility Shift Assay (HMSA).
• HMSA Homogenous Mobility Shift Assay
• Example 5 illustrates the measurement of adalimumab using the methods of this disclosure.
• a standard curve for adalimumab was generated using the methods described herein.
• a TNF-alpha molecule was labeled with a donor fluorophore and an anti- TNF drug Fab fragment was labeled with an acceptor fluorophore.
• TR-FRET occurred in the presence of infliximab.
• the increase in FRET signal of the acceptor is proportional to the amount of drug present in the patients’ sample. The results are tabulated below and shown in FIG. 10.
• Example 5 illustrates a comparison of measuring adalimumab (ADA) using the methods of this disclosure versus Homogenous Mobility Shift Assay (HMSA).
• HMSA Homogenous Mobility Shift Assay

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