Patent Application
20210380721
The presently-disclosed subject matter generally relates to opioid use disorders (OUDs). In particular, certain embodiments of the presently-disclosed subject matter relate compositions and methods for binding opioids while avoiding binding to OUD treatment agents.
Opioid drugs, especially heroin,1 are known as a growing national crisis in America due to the rapidly increasing overdose deaths.2,3 The rapid increase in heroin overdose deaths is related to the fact that heroin is much cheaper and easy to obtain, compared to other opioid drugs. In fact, heroin has become much cheaper than any other drug of abuse, e.g. $10-$20 for a typical single dose (0.1 g) of heroin purchased on the street.4 Notably, heroin itself is actually a prodrug and is converted by cholinesterases' to the highly active metabolites 6-monoacetylmorphine (6-MAM) and morphine, as depicted in
The well-known connection between heroin abuse and prescription opioid abuse is related to the actual availability and costs, in addition to the common brain protein targets (opioid receptors), of these opioid drugs. Indeed, “80% of recent heroin initiates reported that they began their opioid use through the nonmedical use of prescription opioid medications.”4 Those who abuse the prescription drugs most often obtain them from friends and family either through sharing or theft. When they are no longer able to get prescription opioid drugs, they start to use illegal opioid heroin, because heroin is easy to obtain and is relatively in expensive.
Currently used therapeutic agents for treatment of opioid-induced disorders/toxicity include naloxone (a non-selective and competitive antagonist of opioid receptors) used for overdose treatment and buprenorphine (a partial agonist of μ-opioid receptor and an antagonist/partial agonist of many other receptors), methadone (an agonist of μ-opioid receptor, an antagonist of glutamatergic N-methyl-D-aspartate receptor, and a noncompetitive α3β4 neuronal nicotinic acetylcholine receptor), and naltrexone (a competitive antagonist of μ-opioid receptor and other opioid receptors) used for opioid dependence treatment.
These therapeutic agents may be used in various formulations/devices, such as a nasal spray device for naloxone19 for fast toxicity treatment and extended-release naltrexone for relapse-preventing opioid dependence treatment.20 All of these therapeutic agents in current clinical use, and most of other therapeutic candidates under preclinical/clinical development, bind to opioid receptors (and/or related receptors) in the brain and, thus, block/regulate the physiological effects of opioid in the body.
The overdose treatment with naloxone appears to be effective in many cases, however, the naloxone must be introduced relatively quickly following an opioid overdose to revive subject who is overdosing. Further, once overdosed, heroin-dependent users may continue to overdose again and again until a fatal overdose. Some heroin-dependent users survived from one overdose with treatment in a hospital, and then died of another overdose the next day.4 Even worse, the use of naltrexone or its extended-release formulation Vivitrol actually increased heroin overdose.21-23
A truly effective heroin treatment should account for not only rescuing heroin users who have already been overdosed, but also preventing the users from overdose again. In particular, it would be desirable to identify alternative therapeutic strategies to complement the traditional μ-opioid receptor antagonist approach for treatment of heroin-related opioid overdose and dependence.
As alternatives to the traditional μ-opioid receptor antagonist approach, vaccines (that help to elicit antibodies against specific antigens in the body) and monoclonal antibodies (mAbs, for use as the passive vaccination/immunity) have been being developed for treatment of opioid use disorders (OUDs).24-34 A vaccine could be effective for dependency treatment, but would require an immune response to be effective and, thus, would not be useful for overdose treatment. An mAb could be useful for treatment of both the drug dependence and overdose. In particular, an exogenous mAb, which may be used as an exogenous protein therapeutic, is not expected to cross blood-brain barrier (BBB) to interact with any receptors in the brain. Instead, through tightly binding with the opioid drugs in the plasma, the exogenous antibodies are expected to decrease the concentrations of freely available opioids and, thus, attenuate the toxicity and physiological effects of the opioid drugs.
Concerning the feasibility for using an mAb to attenuate the drug toxicity for overdose treatment in addition to the dependence treatment, Janda and associates have demonstrated in mice that an anti-cocaine mAb can be used as an effective antidote to rescue mice after the mice were given a lethal dose of cocaine (post-exposure treatment)35 Based on their encouraging animal data, Janda et al. concluded that “minimal antibody doses were shown to counteract the lethality of a molar excess of circulating cocaine” in the case of the post-exposure treatment and “Passive vaccination with drug-specific antibodies represents a viable treatment strategy for the human condition of cocaine overdose.”35 The work reported by Janda et al. have demonstrated the general concept of utilizing an mAb as a feasible antidote to counteract the drug toxicity post-exposure. Thus, as disclosed herein, it is reasonable to apply a similar concept to an anti-opioid mAb as an antidote to counteract opioid toxicity for overdose treatment, in addition to the dependency treatment.
In fact, there have been various reports of efforts to generate mAbs specifically against morphine25-30,36 or 6-MAM.31-33 Of the anti-morphine mAbs reported so far, a single chain Fv antibody30 can also bind with heroin. However, there has been no demonstration that any of the reported anti-morphine mAbs25-30, 34 can bind with 6-MAM. Conversely, the reported anti-6-MAM mAbs31-33 specifically recognize 6-MAM, but not morphine.
There has been no demonstration that any of the mAbs reported so far25-33, 36 can bind with both 6-MAM (which is mainly responsible for the acute toxicity of heroin) and morphine (which is mainly responsible for the long-term toxicity of heroin), let alone binding with all of the three heroin-related opioids (6-MAM, morphine, and heroin itself).
Interestingly, the 6-MAM-specific mAb (known as 6-MAM-214, with an affinity of 0.3 μM or 300 nM for 6-MAM) was indeed able to reduce the acute heroin effects in mice.31 Thus, as contemplated and described herein, it is possible that a mAb capable of binding with all the three heroin-related opioids (6-MAM, morphine, and heroin itself) would be able to more effectively attenuate the toxicity (including both the acute and long-term toxicity) and physiological effects of heroin.
In general, it is increasingly interesting to develop mAbs as therapeutic proteins. Usually, antibodies may be generated either in vitro, such as the phage and yeast surface display, or in vivo through animal immunization and antibody screening using enzyme-linked immunosorbent assay (ELISA) or Western blot assays, followed by humanization of the identified animal antibody.37
There are a lot of challenges in generating mAbs for therapeutic applications. For example, using the in vivo approach during the antibody discovery stage, immunization affords limited control over antibody affinity and specificity due to the difficulty in controlling antigen presentation to the immune system. Using in vitro methods such as the phage and yeast surface display, a display method is limited by the need of screening a large library etc.37 Hence, it is understandable that none of the mAbs reported so far has demonstrated the ability to bind with all of the three heroin-related opioids (6-MAM, morphine, and heroin itself) or even the ability to bind with both 6-MAM and morphine. Accordingly, there is an unmet need in the art.
The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.
This Summary describes several embodiments of the presently-disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.
The presently-disclosed subject matter includes monoclonal antibodies (mAb) having the ability to bind with one, two, or all three of the heroin-related opioids (6-MAM, morphine, and heroin itself). For example, in some embodiments, the mAb bind with both 6-MAM and morphine. In some embodiments, the mAb binds one or more of the heroin-related opioids, and also does not affect opioid use disorder (OUD) treatment options, such as naloxone and naltrexone. The studies described herein were developed to identify/design an mAb capable of potently binding with all the heroin-related opioids without binding with naloxone and naltrexone.
Also disclosed herein is a general, systematic structure-based virtual screening and design approach for identification of useful antibodies. Exemplary antibodies identified by this process and disclosed herein include UK6Mab01 (a partially humanized antibody) and UK6Mab02 (a fully humanized antibody), which are capable of binding to multiple addictive opioids (including 6-MAM, morphine, heroin, hydrocodone, oxycodone, meperidine, and fentanyl) without significant binding with OUD treatment agents naloxone and naltrexone. Accordingly, antibodies of the presently-disclosed subject matter and as identified by the presently-disclosed methods can serve in connection with treatment of OUDs.
As disclosed herein, the experimental binding affinities reasonably correlate with the computationally predicted binding free energies. Further, the experimental activity data strongly support the computational predictions, establishing that the systematic structure-based virtual screening and humanization design protocol is reliable. The general, systematic structure-based virtual screening and design approach as disclosed herein will be useful for many other antibody selection and design efforts.
The presently-disclosed subject matter includes methods of identifying antibodies that bind an opioid without significant binding with treatment agents for opioid use disorder (OUD). The presently-disclosed subject matter further includes antibodies, and compositions including such antibodies, for use in treating OUD. The presently-disclosed subject matter further includes methods for treating OUD. The presently-disclosed subject matter further includes methods for detecting an opioid in a sample.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which:
SEQ ID NO: 1 is the sequence included in a heavy chain sequence of an antibody, which is set forth in
SEQ ID NO: 2 is the sequence included in a heavy chain sequence of an antibody, which is set forth in
SEQ ID NO: 3 is the sequence included in a heavy chain sequence of an antibody, which is set forth in
SEQ ID NO: 4 is the sequence included in a heavy chain sequence of an antibody, which is set forth in
SEQ ID NO: 5 is the sequence included in a heavy chain sequence of an antibody, which is set forth in
SEQ ID NO: 6 is the sequence included in a light chain sequence of an antibody, which is set forth in
SEQ ID NO: 7 is the sequence included in a light chain sequence of an antibody, which is set forth in
SEQ ID NO: 8 is the sequence included in a light chain sequence of an antibody, which is set forth in
SEQ ID NO: 9 is the sequence included in a light chain sequence of an antibody, which is set forth in
SEQ ID NO: 10 is the sequence included in a light chain sequence of an antibody, which is set forth in
SEQ ID NO: 11 is the sequence included in a light chain sequence of an antibody, which is set forth in
SEQ ID NO: 12 is the sequence included in a light chain sequence of an antibody, which is set forth in
SEQ ID NO: 13 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 14 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 15 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 16 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 17 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 18 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 19 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 20 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 21 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 22 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 23 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 24 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 25 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 26 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 27 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 28 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 29 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 30 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 31 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 32 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 33 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 34 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 35 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 36 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 37 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 38 is the sequence included in a heavy chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 39 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 40 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 41 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 42 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 43 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 44 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 45 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 46 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 47 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 48 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 49 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 50 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 51 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 52 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 53 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 54 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 55 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 56 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 57 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 58 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 59 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 60 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 61 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 62 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 63 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 64 is the sequence included in a light chain sequence of an antibody, which is set forth in Table 1.
SEQ ID NO: 65 is the sequence of a complementarity-determining regions (CDRs) of the heavy chain of an antibody as disclosed herein.
SEQ ID NO: 66 is the sequence of a complementarity-determining regions (CDRs) of the heavy chain of an antibody as disclosed herein.
SEQ ID NO: 67 is the sequence of a complementarity-determining regions (CDRs) of the heavy chain of an antibody as disclosed herein.
SEQ ID NO: 68 is the sequence of a complementarity-determining regions (CDRs) of the heavy chain of an antibody as disclosed herein.
SEQ ID NO: 69 is the sequence of a complementarity-determining regions (CDRs) of the heavy chain of an antibody as disclosed herein.
SEQ ID NO: 70 is the sequence of a complementarity-determining regions (CDRs) of the heavy chain of an antibody as disclosed herein.
SEQ ID NO: 71 is the sequence of a complementarity-determining regions (CDRs) of the light chain of an antibody as disclosed herein.
SEQ ID NO: 72 is the sequence of a complementarity-determining regions (CDRs) of the light chain of an antibody as disclosed herein.
SEQ ID NO: 73 is the sequence of a complementarity-determining regions (CDRs) of the light chain of an antibody as disclosed herein.
SEQ ID NO: 74 is the sequence of a complementarity-determining regions (CDRs) of the light chain of an antibody as disclosed herein.
SEQ ID NO: 75 is the sequence of a complementarity-determining regions (CDRs) of the light chain of an antibody as disclosed herein.
This application contains a sequence listing submitted in accordance with 37 C.F.R. 1.821, named Zhan UKRF 2462 Sequence Listing_ST25.txt, created on Jun. 1, 2021, having a size of 72 KB, which is incorporated herein by this reference.
The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.
The presently-disclosed subject matter includes methods of identifying antibodies that bind an opioid without significant binding with treatment agents for opioid use disorder (OUD).
In some embodiments, the method involves: (a) identifying computationally-refined structures of candidate mAbs using amino-acid sequences of variable domains of the mAbs, homology models of the antibodies, and energy-minimization; (b) superimposing X-ray crystal structures of an opioid with the computationally-refined structures of the candidate mAbs to simulate binding of opioid and antibodies to create opioid-antibody complex structures having one or more binding poses; (c) conducting energy minimization for each binding pose of each opioid-antibody complex structure; (d) calculating binding free energy of each binding pose of each opioid-antibody complex structure; and (e) select the antibodies associated with the binding pose having low binding free energy. In some embodiments, the opioid is one or more of 6-MAM, morphine, heroin, hydrocodone, oxycodone, meperidine, and fentanyl.
In some embodiments, the method also involves determining actual binding affinity of each predicted antibody by preparing and testing each predicted antibody. In some embodiments, the opioid is one or more of 6-MAM, morphine, heroin, hydrocodone, oxycodone, meperidine, and fentanyl.
In some embodiments, the method also involves determining the binding affinity of each predicted antibody with a treatment agent for OUD. In some embodiments, the treatment agent for OUD is naloxone and/or and naltrexone. The presently-disclosed subject matter further includes antibodies, and compositions including such antibodies, for use in treating OUD. In some embodiments, the antibody is discovered by a method as disclosed herein. In some embodiments, the antibody is UK6Mab01 or UK6Mab02.
The presently-disclosed subject matter includes an isolated antibody or antigen fragment thereof that binds one or more of 6-MAM, morphine, heroin, hydrocodone, oxycodone, meperidine, and fentanyl, and does not bind naloxone or naltrexone. In some embodiments, the antibody is selected from the group consisting of: (a) an antibody comprising the sequence of SEQ ID NO: 5; (b) an antibody comprising the sequence of SEQ ID NO: 12; (c) an antibody comprising the sequence of SEQ ID NOs: 5 and 12; (d) an antibody comprising the sequence of SEQ ID NO: 25; (e) an antibody comprising the sequence of SEQ ID NO: 51; (f) an antibody comprising the sequence of SEQ ID NO: 25 and 51; (g) an antibody comprising the sequence of (i) SEQ ID NO: 65, 66, or 67, and (ii) SEQ ID NO: 68, 69, or 70; (h) an antibody comprising the sequence of (i) SEQ ID NO: 71 or 72, (ii) GTN, STN, or DTS, and (iii) SEQ ID NO: 73, 74, or 75; and (i) an antibody comprising the sequence of (i) SEQ ID NO: 65, 66, or 67, (ii) SEQ ID NO: 68, 69, or 70, (iii) SEQ ID NO: 71 or 72, (iv) GTN, STN, or DTS, and (v) SEQ ID NO: 73, 74, or 75. In some embodiments, the antibody comprises a sequence selected from the group consisting of the sequence of any one of SEQ ID NOS: 1-75.
The presently-disclosed subject matter further includes methods for treating OUD, which involve administering an antibody as disclosed herein to a subject in need thereof.
The presently-disclosed subject matter further includes methods for detecting an opioid in a sample, which includes contacting the sample with an antibody as disclosed herein, and detecting binding of the antibody to a ligand.
While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.
All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.
Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9):1726-1732).
Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.
In certain instances, nucleotides and polypeptides disclosed herein are included in publicly-available databases, such as GENBANK® and SWISSPROT. Information including sequences and other information related to such nucleotides and polypeptides included in such publicly-available databases are expressly incorporated by reference. Unless otherwise indicated or apparent the references to such publicly-available databases are references to the most recent version of the database as of the filing date of this application.
The present application can “comprise” (open ended) or “consist essentially of” the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.
Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.
As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, in some embodiments ±0.1%, in some embodiments ±0.01%, and in some embodiments ±0.001% from the specified amount, as such variations are appropriate to perform the disclosed method.
As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.
An “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable domain of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen-binding portion thereof that competes with the intact antibody for specific binding, fusion proteins comprising an antigen-binding portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. Antigen-binding portions include, for example, Fab, Fab′, F(ab′)2, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelid antibodies), portions including complementarity determining regions (CDRs), single chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes (i.e., isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (subtypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
The terms “antigen-binding portion” or “antigen-binding fragment” of an antibody (or simply “antibody portion”), as used interchangeably herein, refers to one or more portions of an antibody that retain the ability to specifically bind to an antigen (e.g., delta-CN). It has been shown that the antigen-binding function of an antibody can be performed by portions of a full-length antibody.
A “variable domain” of an antibody refers to the variable domain of the antibody light chain (VL) or the variable domain of the antibody heavy chain (VH), either alone or in combination. As known in the art, the variable domains of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs) also known as hypervariable regions, and contribute to the formation of the antigen-binding site of antibodies. If variants of a subject variable domain are desired, particularly with substitution in amino acid residues outside a CDR (i.e., in the framework region), appropriate amino acid substitution, in some embodiments, conservative amino acid substitution, can be identified by comparing the subject variable domain to the variable domains of other antibodies which contain CDR1 and CDR2 sequences in the same canonical class as the subject variable domain.
As used herein, “monoclonal antibody” refers to an antibody 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. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues.
The term “antigen (Ag)” refers to the molecular entity used for immunization of an immunocompetent vertebrate to produce the antibody (Ab) that recognizes the Ag or to screen an expression library (e.g., phage, yeast or ribosome display library, among others). Herein, Ag is termed more broadly and is generally intended to include target molecules that are specifically recognized by the Ab, thus including portions or mimics of the molecule used in an immunization process for raising the Ab or in library screening for selecting the Ab.
Generally, the term “epitope” refers to the area or region of an antigen to which an antibody specifically binds, i.e., an area or region in physical contact with the antibody. Thus, the term “epitope” refers to that portion of a molecule capable of being recognized by and bound by an antibody at one or more of the antibody's antigen-binding regions.
An antibody that “preferentially binds” or “specifically binds” or “selectively bind” (used interchangeably herein) to an epitope is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art. A molecule is said to exhibit “specific binding,” “selective binding,” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. Also, an antibody “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration to that target in a sample than it binds to other substances present in the sample. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding.
As used herein, “pharmaceutically acceptable carrier” or “pharmaceutical acceptable excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. In some embodiments, diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods.
The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention.
Antibodies, including a total of five classes, i.e. immunoglobulin (Ig)A, IgD, IgE, IgG, and IgM, are known as affinity proteins that are a key component of the adaptive immune system.37 The ability of antibodies to bind to foreign invaders with high affinity and specificity is central to their functions. IgGs, including four subclasses, i.e. IgG1 to 4, are the most abundant class of antibodies, constituting approximately 75% of the serum immunoglobulin repertoire. Notably, all IgGs share the same overall architecture, with differences only in six loops known as the complementarity-determining regions (CDRs) in the antigen-binding site.
Utilizing these remarkable structural features, the general strategy of the systematic structure-based virtual screening approach to antibody selection and identification as disclosed herein starts from collection and structural modeling of all mAbs whose amino-acid sequences (particularly the six CDRs) are available, with a view toward identifying the available mAbs can meet the above-mentioned goal—capable of potently binding with all the heroin-related opioids without binding with naloxone and naltrexone. Using the modeled structure of each mAb available, one can dock each of the interested ligands (i.e. 6-MAM, heroin, morphine, naloxone, and naltrexone in the current study) to the antigen-binding site and carry out further simulations and calculations to computationally estimate the binding free energy with each ligand. Based on the computationally estimated binding free energies, one can predict which mAb most likely can meet the goal of the potency and selectivity.
Specific for the need of binding with multiple opioids, the computationally estimated binding free energies clearly indicated which mAb should have the highest overall potency for binding with 6-MAM, morphine, and heroin without significant binding affinity to naloxone and naltrexone. The computationally selected most promising mAb was modeled further for humanization, and the predicted humanized mAb was prepared and tested in vitro for its actual binding affinities with various ligands including 6-MAM, heroin, morphine, naloxone, and naltrexone etc. Described below are the detailed procedures of the used computational methods.
The amino-acid sequences of variable domains of the antibodies were obtained from the IMGT/LIGM-DB database (imgt.org)38 or manually collected from the references cited (see Tables 1 and 2). The homology models of antibodies were built using the PIGSpro software39 which includes known X-ray crystal structures of a number of antibodies. Using the software, for homology modeling of each antibody structure, the template (used for the framework structure modeling) was selected based on the sequence alignment with those of the known X-ray crystal structure, the loops were kept in the similar canonical structure of template for the loop modeling method, and all other parameters were set as the default of the software.
Then the initial homology models were refined by performing a series of energy minimization processes. Specifically, the Amber14SB force field40 was applied for the proteins in vacuo using AmberTools18.41 The nonbonded cutoff for the real-space interactions was set to 10 Å. Two stages of energy minimization were conducted using a hybrid protocol of 8000 steps of steepest descent minimization followed by a conjugate gradient minimization until the convergence criterion (the root-mean-square of the energy gradient is less than 1.0×10−4 kcal/mol·Å) was satisfied or the maximum of 2000 iteration steps was reached. During the energy minimization, a force constant of 10 kcal/mol·Å2 was applied on the antibody backbone atoms. Then the final conformations were used for virtual screening described below.
The computationally refined antibody structures were superimposed with the X-ray crystal structure of the complex of morphine with antibody 9B134 (PDB: 1Q0Y) using the PyMol software42 in order to transform the atomic coordinates of all proteins into the same coordinate system with a commonly defined center of box required to define for systematic molecular docking, and the MglTools43 software was used to prepare the protein and ligand pdbqt files for the docking. During the docking, the binding site was determined based on the antibody-morphine complex in 9B1 (PDB: 1Q0Y), the geometric center of the co-crystallized antibody-morphine complex was indicated as the active center of the docking box (the size_x, size_y, and size_z were set to 30, 30, and 30, respectively) which was large enough to cover the entire region of the binding site.
The docking calculations were conducted by using the AutoDock Vina software,43 and all the default parameters were adopted. For each antibody-ligand complex, the top-4 ranked binding poses of the complex were selected for further computational evaluation in multiple steps.
First, the selected complex structures were energy-minimized using the same approach as described above for the energy-minimization of antibody structures without a ligand. The general Amber force field (gaff)44 was used for the ligands. Second, the energy-minimized complex structure was relaxed by performing a short (20 ps) molecular dynamics (MD) simulation using the SANDER module of the AmberTools18 software41 in vacuo with a constant temperature (T=300 K). A restrain (with a force constant of 2 kcal/mol·Å2) was applied on the backbone of antibody. The SHAKE algorithm45 was used to restrain the covalent bonds with hydrogen atoms, and the time step for the MD simulation was set to 2 fs. The long-range electrostatic interactions were treated by using the particle mesh Ewald (PME) algorithm,46 and the nonbonded cutoff for the real-space interactions was set to 12 Å. Third, the last snapshots of the MD simulations were energy-minimized again using the same method described above for the energy-minimization of the free antibody structures without a ligand. Finally, the MMPBSA module of the AmberTools18 software was used to calculate the binding free energy of each antibody-ligand binding pose, leading to the identification of the antibody-ligand binding pose associated with the lowest binding free energy for each antibody-ligand complex.
With the lowest binding free energy pose for each antibody-ligand pair and the corresponding binding free energy determined, the antibody was selected with the lowest possible binding free energy (i.e. the highest possible binding affinity) with 6-MAM and with the best possible overall binding affinities with other heroin-related opioids as well as the desirable selectivity over naloxone and naltrexone.
Humanization of the computationally selected antibody (i.e. murine antibody Ab13 in this study) was performed by using the X-ray crystal structure of Ab13 complexed with morphine34 (PDB: 1Q0Y) from PDB database (rcsb.org/pdb/home/home.do) and the immunoinformatic modelling tools made available by the IMGT database.38 Briefly, the variable heavy and light chain sequences (VH and VL) of the murine antibody were compared with human germline sequences using the IMGT/DomainGapAlign tools.38 The top-ranked human germline sequence was used as the template. Murine antibody residues that differ from the human sequences on the surface area were replaced, excluding the residues near the binding pocket and anchor residues. Individual residues that are clearly not involved in the binding with the ligands in the murine antibody were changed to the corresponding residues of the human antibody. The image of the aligned sequences was created using the ESPript 3.0 software.47
The above-mentioned sequence modification based on the sequence alignment and simple structural modeling led to multiple (three) possible choices of the sequence of the humanized antibody, i.e. three possible humanized antibodies denoted as H1L1, H1L2, and H1L3. The initial structures of the antibodies (generated by using the PIGSpro software39 as described above) were refined further by performing a series of energy minimization processes and restrained MD simulations in order to know which one of the three choices is most reasonable. Concerning the computational details, the Amber14SB force field40 and the generalized Amber force field (gaff)44 were used for the proteins and ligands, respectively. The TIP3P water molecules48 were added as the solvent and the solute atoms were at least 10 Å away from the boundary of the water box using AmberTools18.41 The counterions (i.e. Na+ ions for murine antibodies or Cl− ions for humanized antibodies) were added to neutralize the system. The long-range electrostatic interactions were handled by the particle mesh Ewald (PME) algorithm,46 and the nonbonded cutoff for the real-space interactions was set to 10 Å.
Energy minimization was performed using a hybrid protocol of 8000 steps of the steepest descent energy-minimization followed by the conjugate gradient energy-minimization until the convergence criterion (the root-mean-square of the energy gradient is less than 1.0×10−4 kcal/mol·Å) was satisfied or the maximum of 2000 iteration steps was reached. During the energy minimization, a force constant of 100 kcal/mol·Å2 was applied on the ligand and protein backbone atoms. Then the systems were heated up from 0 to 303.15 K linearly over a time period of 50 ps with the restraint (force constant of 10 kcal/mol·Å2) on all heavy atoms in the NVT ensemble, followed by equilibrating for 325 ps with a Langevin thermostat49 in the NPT (P=1 atm and T=303.15 K) ensemble by gradually decreasing the force constant from 10 to 0.2 kcal/mol·Å2. Finally, the 5-ns production run was carried out with the PMEMD module of the Amber12 in the NPT (P=1 atm and T=303.15 K) ensemble. The SHAKE algorithm was used to restrain the covalent bonds with hydrogen atoms, and the time step was set to 2 fs, the snapshots were saved every 2 ps. The RMSD values were calculated by CPPTRAJ module of AmberTools18 using the energy-minimized conformations as the references.
To construct the chimeric antibody (UK6Mab01) and the (fully) humanized antibody (UK6Mab02), the amino acid sequences of heavy and light chains of variable domains of UK6Mab01 and UK6Mab02 were linked with human immunoglobulin heavy constant gamma 1 (IgG1, P01857, heavy chain) and human immunoglobulin kappa constant (IGKC, P01834, light chain), respectively.
The heavy and light chains were translated to human gene sequences by using the Backtranseq provided by the EMBL-EBI,50 and the codon was optimized using the COOL.51 The Kozak sequence and signal peptide sequences for heavy chain or light chain were added to optimized genes. Then the genes for heavy chain and light chain were linked by inserting an Internal Ribosome Entry Site (IRES) between them. The designed genes were synthesized by GeneArt (Invitrogen, Carlsbad, Calif.), followed by cloning the genes into the pCMV-MCS vector at the BamHI and SalI sites for the humanized antibody, and at the BamHI and XhoI sites for the chimeric antibody. The oligonucleotides were synthesized by the Eurofins Genomics (Louisville, Ky.), restriction enzymes and the KLD Enzyme Mix used for ligation were purchased from New England Biolabs (Ipswich, Mass.). The final plasmids used for transfection were verified by sequencing services provided by Eurofins Genomics (Louisville, Ky.).
CHO-S cells were grown under the condition of 37° C. and 8% CO2 in a humidified atmosphere. The constructed expression vectors for the chimeric and humanized antibodies were transfected into CHO-S cells using Minis TransIT-PRO® Transfection Kit. 400 mL cells at the density of 2×106 cells/mL were transfected with 400 μg of expression vector and 400 μL of transfection reagent. Culture supernatants were harvested 5 days after transfection by centrifugation with 10000 rpm for 15 min at 4° C. Antibodies were purified with using a protein A resin (Mab Select SuRe™ ordered from GE Healthcare, Chicago, Ill.), as used previously.52-54 Briefly, 5 mL resin was packed in a column, equilibrated with 20 mM Tris-Cl (pH=7.4), loaded the culture supernatants with flow rate of 1-2 mL/min, washed with wash buffer (20 mM Tris-Cl, 300 mM NaCl, pH=7.4), and eluted with elution buffer (50 mM citric acid, 300 mM NaCl, pH=4). Then the eluate was concentrated and stored in PB buffer. Purified proteins were analyzed by SDS-PAGE (Invitrogen, Carlsbad, Calif.).
The binding constant of the antibody with [H3]-morphine were tested using liquid scintillation counting. Briefly, 2 nM [H3]-morphine was incubated with different concentration of antibody at room temperature for 60 minutes. The total volume of mixture was 100 and pH was 7.4. Following filtration with EMD Millipore Amicon™ Ultra-0.5 Centrifugal Filter (30 kD) and EMD Millipore Amicon™ Ultra 0.5 mL vials, 50 μL of the filtrate was added to 3 mL of 3a70BTM complete counting cocktail (RPI Research Products, Mount Prospect, Ill.). After vortex, the radioactive value of the cocktail was read, the Kd value was calculated using the GraphPad Prism 7 software.
The binding affinities of each antibody with other drugs were calculated from its binding constant with [H3]-morphine (Kd) and IC50 values against corresponding drugs, using the IC50-to-Ki converter software (umich.edu/˜shaomengwanglab/software/calc_ki/index.html>. The IC50 values of corresponding drugs were measured as follows. 100 μL of mixture (pH 7.4) containing 2 nM [H3]-morphine, 20 or 60 nM of the antibody, and a varying concentration of drug was incubated at room temperature for 60 minutes. Then the mixture was filtered with EMD Millipore Amicon™ Ultra-0.5 Centrifugal Filter (30 kD) and EMD Millipore Amicon™ Ultra 0.5 mL Vials, 50 μL of the filtrate was transferred to 3 mL of 3a70BTM complete counting cocktail (RPI Research Products, Mount Prospect, Ill.), then the radioactive value of the cocktail was read, and the IC50 value was calculated using the GraphPad Prism 7 software.
According to the virtual screening of available antibodies, including all of those listed in Table 1 with known experimental binding affinity to any of the heroin-related opioids, 6-MAM may potentially bind with the 26 known antibodies listed in Table 1, but with different binding free energies ranging from −16.6 kcal/mol (for the most potent one, i.e. Ab13) to −2.0 kcal/mol (which represents a negligible binding affinity) (see Table 2). Hence, the binding free energies of these 26 antibodies with morphine, heroin, naloxone, and naltrexone were computationally estimated. All the calculated binding free energies are summarized in Table 2 in comparison with available experimental data.
YANWVQEKPDHLFTGL
LPGTGRTFYSENFKVIC
LVFGGGTKLTVL
aThe experimental activity data were collected from the references cited. Reported Ka and IC50 values were coverted to the corresponding Kd and Ki, respectively.
bnd (not determined) means no available experimental data.
1Jun-ichi, S., et al. (1993).
2Glasel, J.A., et al. (1983).
3Pozharski, E., et al. (2004).
4Kussie, P.H., et al. (1991).
5Moghaddam, A., et al. (2003).
6Matsukizono, M., et al. (2013).
Interestingly, for many of these antibodies listed in Table 2, experimental binding free energies (converted from the previously reported Kd, Ki, or IC50 values according to the well-known thermodynamic equation) have already been known for their binding with morphine and naloxone. According to the experimental binding affinities summarized in Table 2, the most potent anti-morphine antibodies (Ab1 to 9), Ab15, and Ab16, can also potently bind to naloxone with nanomolar Kd or IC50 values (ranging from 33 nM to 145 nM). Ab15 even can potently bind with naltrexone (IC50=310 nM). A therapeutic antibody capable of binding with naloxone or naltrexone would be problematic because it would block the favorable pharmacologic action of naloxone (the currently available therapeutic agent for opioid overdose treatment) or naltrexone (the currently available therapeutic agent for opioid dependence treatment). For this reason, these potent anti-morphine antibodies would not be suitable for consideration as the desirable therapeutic candidates. Nevertheless, the available experimental data for enough number of antibodies binding with morphine and naloxone allowed us to analyze the potential correction between and computational and experimental data and, thus, validate the computational protocol. In comparison, only six antibodies have experimental data available for their binding with 6-MAM, with a very narrow range of the Kd values (100 to 300 nM) that had limited the correlation analysis for 6-MAM before further experimental data was obtained to expand the range (see below).
As showed in
Within all antibodies listed Table 2, Ab13 was predicted to have the lowest binding free energy (−15.6 kcal/mol) with 6-MAM; the lowest binding free energy means the highest binding affinity. Ab13 was also predicted to have high binding affinities with morphine and heroin (corresponding to the calculated binding free energies of −15.1 and −16.6 kcal/mol, respectively). Interestingly, Ab13 was predicted to have the highest binding free energy (i.e. the lowest binding affinity) with naloxone and naltrexone (corresponding to the calculated binding free energies of −1.8 and −3.2 kcal/mol, respectively). In other words, Ab13 was predicted to be the most promising antibody capable of potently binding with 6-MAM, morphine, and heroin without significant binding affinity with naloxone and naltrexone.
To identify the key residues of Ab13 for the binding with 6-MAM, morphine, and heroin, the X-ray crystal structure of the complex of Ab13 with morphine was taken from the RC SB Protein Data Bank, together with the predicted binding structures of 6-MAM and heroin, were analyzed and presented in
In addition, as shown in
Ab13 was acquired from mouse by using the hybridoma technique.34 To make use of this antibody for future use in human, Ab13 must be humanized to reduce immune response. Currently, many methods have been used in antibody humanization, the most wildly used methods are complementarity determining regions (CDRs) graft and antibody resurfacing.55 As CDRs graft change the framework regions (FRs) of the humanized antibody, certain key residues in FRs of Ab13 play an important role in binding with 6-MAM, heroin, and morphine according to the modelled binding structures (see
Initially, the amino-acid sequences of the variable domains of the heavy (VH) and light (VL) chains of Ab13 were analyzed using the IMGT/DomainGapAlign tools,38 and the closest human germline V and J genes were identified. As shown in
Further, for assessment of the structural stability of the humanized antibody within the above three possible choices of the humanized antibody, MD simulations were carried out to examine the fluctuation of the residues in the binding pocket and the backbone atoms of the three possible humanized antibodies (denoted as H1L1, H1L2, and H1L3). For comparison, MD simulations were also performed on the original structures of Ab13 and its complex with morphine, and the time-dependent root-mean-square deviations (RMSDs) for all the simulated structures are provided in
In comparison, the pocket residues of H1L1 were stable during the MD simulation on H1L1 without morphine bound, but less stable during the MD simulation on the H1L1-morphine complex. On the contrary, the pocket residues of H1L2 were less stable during the MD simulation on H1L2 without morphine bound, as the RMSDs continually increased during the simulation, although the simulated H1L2-morphine complex structure was relatively more stable. Within the three humanized antibodies, H1L3 has the most stable structure, as reflected by the low RMSDs (for both the pocket residues and backbone atoms, as well as morphine bound) that were stable during the MD simulations on H1L3 with or without morphine bound. So, H1L3 was the final choice of the fully humanized antibody.
Besides, for the purpose of functional comparison, a chimera antibody (a partially humanized antibody) was also designed, in which only the Fc part of the mouse Ab13 was replaced by the Fc of human IgG-1. Such a chimera antibody was expected to have the same functions with the mouse Ab13. For convenience, the chimera antibody and the fully humanized antibody (H1L3) are denoted as UK6Mab01 and UK6Mab02, respectively, in the discussion below.
To prepare the designed antibodies UK6Mab01 and UK6Mab02, the sequences of heavy and light chains were assembled with human immunoglobulin heavy constant gamma 1 (IgG1, P01857) and human immunoglobulin kappa constant (IGKC, P01834), respectively. The use of internal ribosome entry site (IRES) allowed for the co-expression of a light chain and its corresponding heavy chain under the control of the same promoter. Both UK6Mab01 and UK6Mab02 proteins were expressed in the CHO-S cells and purified. A major band of approximately 75 kDa was observed, corresponding to the integrity of heavy and light chains. Two other major bands with molecular masses of approximately 50 kDa (heavy chain) and 25 kDa (light chain) were also observed (
The binding affinities of UK6Mab01 and the UK6Mab02 with various ligands were assessed by the liquid scintillation counting method. Both the UK6Mab01 and UK6Mab02 were able to potently bind to morphine with a Kd value of 33.7 nM (
a Data from reference 33.
b nd (not determined) means no available experimental data.
In addition, UK6Mab01 and UK6Mab02 were examined to determine whether they can potently bind to other drugs including naloxone, naltrexone, hydrocodone, meperidine, oxycodone, methadone, amethaphetamine, ketamine, cocaine, and fentanyl (
Further, with the newly obtained binding affinity of 6-MAM with UK6Mab01, the computationally predicted binding free energies with 6-MAM were also able to be shown to excellently correlate with the corresponding experimental data (
The systematic structure-based virtual screening of available monoclonal antibodies and computational design of antibody humanization have led to discovery of promising antibodies, including the partially humanized antibody UK6Mab01 and the fully humanized antibody UK6Mab02, that can potently bind to multiple addictive opioids (including 6-MAM, morphine, heroin, and hydrocodone) without significant binding with currently available opioid overdose/dependence treatment agents naloxone and naltrexone. Specific for UK6Mab01, it was determined that Kd=4.8 nM for 6-MAM, 1.3 nM for heroin, 33.7 nM for morphine, 8.2 nM for hydrocodone, and 2.7-32.9 μM for oxycodone, meperidine, and fentanyl, without significant binding affinity to other drugs tested. For UK6Mab02, it was determined that Kd=11.9 nM for 6-MAM, 11.8 nM for heroin, 127 nM for morphine, 39.3 nM for hydrocodone, and 6.9-15.7 μM for oxycodone, fentanyl, and meperidine, without significant binding affinity to other drugs tested. The fully humanized antibody are contemplated for use in treatment of OUDs.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, including the references set forth in the following list:
It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.
This application claims priority from U.S. Provisional Application Ser. No. 63/032,995 filed Jun. 1, 2020, the entire disclosure of which is incorporated herein by this reference.
This invention was made with government support under grant number CHE-1111761 awarded by the National Science Foundation (NSF). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63032995 | Jun 2020 | US |
