Patent Application
20220267416
This disclosure relates to binding agents with specificity for human immunodeficiency virus (HIV), methods for making the same, and to methods for using the same to treat and/or prevent HIV infection.
As we enter the fourth decade of the HIV epidemic, significant advances have been made in the understanding of HIV pathogenesis and in the development of potent and safe antiviral drugs. More than 30 antiviral drugs have been registered and the impact of combination antiretroviral therapy (ART) on both morbidity and mortality has been remarkable. However, despite the long-term suppression of HIV replication achieved in patients with optimal adherence to ART, HIV invariably rebounds after interruption of therapy. Furthermore, successful therapy does not induce or allow restoration/development of virus-specific immune responses capable of controlling HIV replication in the absence of ART. Thus, life-long ART is needed to control HIV replication and associated disease in the large majority of HIV infected subjects.
A number of immunological interventions have been investigated in the past and currently being further developed with the goal to achieve HIV functional cure, wherein viral replication is suppressed without sustained antiviral therapy. Therapeutic vaccine strategies have been the primary intervention strategy investigated but the results have shown modest efficacy in experimental animal models and patients with the exception of a CMV-based vector HIV vaccine (50% efficacy in the NHP model). Recent studies have generated interesting results on the possibility of using anti-envelope broad neutralizing antibodies (bNabs) as therapeutic agents in HIV infection.
There is a need in the art for additional reagents for targeting HIV, especially neutralizing antibodies, and methods for using the same. This disclosure addresses those needs by providing reagents and methods that may be used to target HIV and cells and/or tissues infected by and/or harboring the same.
In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.
Exemplary LN02 variable light chain amino acid sequences.
Exemplary LN02 variable heavy chain amino acid sequences.
This disclosure relates to binding agents with specificity for human immunodeficiency virus (HIV), methods for producing such binding agents, as well as methods for using such binding agents to treat, prevent and/or ameliorate HIV infection. In some embodiments, this disclosure provides a binding agent(s) comprising a variable region shown in
This disclosure relates to binding agents having binding affinity for human immunodeficiency virus (HIV). In some embodiments, the binding agent can bind HIV antigens on viral particles per se or on the surface of cells in vitro and/or in vivo. The binding agents may also bind isolated HIV antigens and/or fragments and/or derivatives thereof, typically in vitro. Also provided are methods for using such binding agents to diagnose, treat, prevent and/or ameliorate one or more diseases associated with HIV. For instance, the binding agents may be antibodies (e.g., monoclonal antibodies) that may react with and/or bind to the epitopes of HIV or polypeptides thereof. The binding agents may be useful for treating disease caused by HIV, such as Acquired Immune Deficiency Syndrome (AIDS). In some embodiments, the binding agents described herein may selectively target and/or eliminate HIV and/or HIV-infected cells containing HIV (e.g., replication competent HIV) and/or expressing proteins thereof. In some embodiments, such cells may be reservoirs for replication competent HIV. In some embodiments, binding agents having, for instance, different specificities (e.g., recognizing different epitopes) may be combined to HIV activity such as infection, replication and/or spread to other cells. In some embodiments, the binding agents described herein may also provide for the selective elimination and/or suppression of HIV or HIV-expressing cells. In some embodiments, the binding agents described herein may be used to suppress and/or eliminate HIV and/or HIV-expressing cells to treat, for instance, HIV infection and/or AIDS. Other embodiments, uses and the like are described below.
The binding agents may be antibodies such as monoclonal antibodies. As shown in the examples herein, the techniques discussed below have been used to identify a fully human mAb termed “LN02”, having particular amino acid sequences and characteristics that have described here (e.g.,
Exemplary, and preferred, amino acid sequences of the three heavy chain CDRs (CDRH1, CDRH2, CDRH3), three light chain CDRs (CDRL1, CDRL2, CDRL3), VH and VL domains of the LN02M binding agents (e.g., antibodies) are summarized below and in
In some embodiments, the LN02M binding agent comprises a modified LN02 CDRH1 (YGSISRHFWG), corresponding to amino acids 26-35 of the LN02_VH amino acid sequence illustrated in
In some embodiments, the LN02M binding agent comprises a modified LN02 CDRH2 (HMHHLGVKYVNPSLK), corresponding to amino acids 50-64 of the LN02_VH amino acid sequence illustrated in
In some embodiments, the LN02M binding agent comprises a modified LN02 CDRH3 (VRMGARMSDIAFFSFGD), corresponding to amino acids 96-112 of the LN02M_VH amino acid sequence illustrated in
In some embodiments, the LN02M binding agent comprises a modified LN02 CDRL1 (WGYYMGSKPVN), corresponding to amino acids 23-33 of the LN02M_VL sequence illustrated in
In some embodiments, the LN02M binding agent comprises a modified LN02 CDRL2 (YDDERDS), corresponding to amino acids 49-55 of the LN02M_VL sequence illustrated in
In some embodiments, the LN02M binding agent comprises a modified LN02 CDRL3 (QVWDSKYEEIY), corresponding to amino acids 88-98 of the LN02M_VL sequence illustrated in
In some embodiments, an LN02M binding agent can comprise modifications to the LN02 variable heavy and/or variable light chain regions amino acid sequences outside the CDRs, as those CDRs are illustrated in
In some embodiments, an LN02M binding agent can comprise an LN02M polypeptide modified as illustrated in
A binding agent of this disclosure can comprise any of the modified CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences described above, or conservatively substituted variants thereof. Such a binding agent may be a polypeptide, such as an antibody, as described in more detail below.
A binding agent of this disclosure may comprise, for example, any one or more of the amino acid sequences (i.e., polypeptide sequences) of SEQ ID NOS. 3-92, SEQ ID NOS. 95-233, and/or those shown in
Combinations of LN02 CDRs with LN02M CDRs, as well as modified amino acid sequences outside of CDRs, can be combined with one another as desired, typically while maintaining the functional characteristics such as HIV pseudo-virus neutralization (as illustrated by Tables 1-3 and 7-14,
In some embodiments, the binding agent can be a monoclonal antibody (mAb) or a fragment or derivative thereof. In some embodiments, the binding agent may be an HIV-binding fragment of such a monoclonal antibody (mAb). In some embodiments, one or more LN02M CDR(s), and/or an amino acid sequence comprising such CDR(s), and in some embodiments other modified sequences present outside the CDR regions (see, e.g.,
It is preferred that the binding agent (e.g., antibody, or the antigen binding fragment thereof), comprises one or more amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to at least one LN02M variable region and/or CDR and/or non-CDR amino acid sequences (e.g., the modified CDRs illustrated in
The LN02M variable region and/or LN02M CDR and/or non-CDR amino acid sequences may be used in combination with one or more other variable region/CDR amino acid sequences available to those of ordinary skill in the art. Such variable region/CDR amino acid sequences may alternatively and/or also be adjoined to one or more types of constant region polypeptides of an antibody molecule. For instance, the LN02M CDRs can be adjoined to or associated with the constant regions of any antibody molecule of the same or a different species (e.g., human, goat, rat, sheep, chicken) and/or antibody subtype of that from which the CDR amino acid sequence was derived. For instance, an exemplary binding agent LN02M may be, or may be derived from, one having about the same neutralizing activity and/or binding the same or similar epitopes and/or exhibiting about the same affinity as another binding agent comprising one or more LN02M variable region and/or CDR and/or non-CDR amino acid sequences. The binding agent may comprise an antibody heavy and/or a light chain that each comprises one or more constant and/or variable regions. Any of the amino acid sequences described herein (e.g., the LN02M variable region and/or CDR and/or non-CDR amino acid sequences), and/or any fragments and/or derivatives thereof, may also be combined with any other variable region and/or CDR in any order and/or combination to form new binding agents, e.g., hybrid and/or fusion binding agents, and/or inserted into other heavy and/or light chain variable regions using standard techniques.
This disclosure also provides for the use of such binding agents to isolate, identify, and/or target HIV and/or cells harboring and/or infected by HIV and/or expressing HIV antigens. In certain embodiments, such binding agents may be reactive against HIV antigens such as proteins expressed on the surface of cells. In some embodiments, the binding agent(s) is an antibody (antibodies). The term “antibody” or “antibodies” may refer to whole or fragmented antibodies in unpurified or partially purified form (e.g., hybridoma supernatant, ascites, polyclonal antisera) or in purified form. The antibodies may be of any suitable origin or form including, for example, murine (e.g., produced by murine hybridoma cells), or expressed as humanized antibodies, chimeric antibodies, human antibodies, and the like. For instance, antibodies may be wholly or partially derived from human (e.g., IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1 and IgA2), IgD, and IgE), canine (e.g., IgGA, IgGB, IgGC, IgGD), chicken (e.g., IgA, IgD, IgE, IgG, IgM, IgY), goat (e.g., IgG), mouse (e.g., IgG, IgD, IgE, IgG, IgM), pig (e.g., IgG, IgD, IgE, IgG, IgM), and/or rat (e.g., IgG, IgD, IgE, IgG, IgM) antibodies, for instance. Methods of preparing, utilizing and storing various types of antibodies are well-known to those of skill in the art and would be suitable in practicing the present invention (see, for example, Harlow, et al. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Harlow, et al. Using Antibodies: A Laboratory Manual, Portable Protocol No. 1, 1998; Kohler and Milstein, Nature, 256:495 (1975)); Jones et al. Nature, 321:522-525 (1986); Riechmann et al. Nature, 332:323-329 (1988); Presta (Curr. Op. Struct. Biol., 2:593-596 (1992); Verhoeyen et al. (Science, 239:1534-1536 (1988); Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991); Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991); Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995); as well as U.S. Pat. Nos. 4,816,567; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and, 5,661,016). In certain applications, the antibodies may be contained within hybridoma supernatant or ascites and utilized either directly as such or following concentration using standard techniques. In other applications, the antibodies may be further purified using, for example, salt fractionation and ion exchange chromatography, or affinity chromatography using Protein A, Protein G, Protein A/G, and/or Protein L ligands covalently coupled to a solid support such as agarose beads, or combinations of these techniques. The antibodies may be stored in any suitable format, including as a frozen preparation (e.g., −20° C. or −70° C.), in lyophilized form, or under normal refrigeration conditions (e.g., 4° C.). When stored in liquid form, for instance, it is preferred that a suitable buffer such as Tris-buffered saline (TBS) or phosphate buffered saline (PBS) is utilized. In some embodiments, the binding agent may be prepared as an injectable preparation, such as in suspension in a non-toxic parenterally acceptable diluent or solvent. Suitable vehicles and solvents that may be utilized include water, Ringer's solution, and isotonic sodium chloride solution, TBS and/or PBS, among others. Such preparations may be suitable for use in vitro or in vivo may be prepared as is known in the art and the exact preparation may depend on the particular application.
The binding agents described herein are not, however, in any way limited to antibodies (i.e., whole antibodies). For example, the binding agent may be any compound exhibiting similar binding properties as another (e.g., a mimetic). For example, an exemplary binding agent may be one that binds HIV and/or can compete with another binding agent having specificity therefor (e.g., a monoclonal antibody such as a LN02M antibody). In some embodiments, the mimetic may exhibit substantially the same affinity in binding assays as the binding agent (e.g., monoclonal antibody) to which it is being compared. The affinity a particular binding agent may be measured by any suitable assay including but not limited to FACS staining of cell surface HIV antigens (e.g., polypeptides). One binding agent may be said to have “substantially the same affinity” as another where the measurements (e.g., nm) are within about any of 1-20, 1-5, 5-10, 10-15, or 15-20 percent of one another. Exemplary mimetics may include, for example, organic compounds that specifically bind HIV, or an affibody (Nygren, et al. FEBS J. 275 (11): 2668-76 (2008)), affilin (Ebersbach, et al. J. Mol. Biol. 372 (1): 172-85 (2007)), affitin (Krehenbrink, et al. J. Mol. Biol. 383 (5): 1058-68 (2008)), anticalin (Skerra, A. FEBS J. 275 (11): 2677-83 (2008)), avimer (Silverman, et al. Nat. Biotechnol. 23 (12): 1556-61 (2005)), DARPin (Stumpp, et al. Drug Discov. Today 13 (15-16): 695-701 (2008)), Fynomer (Grabulovski, et al. J. Biol. Chem. 282 (5): 3196-3204 (2007)), Kunitz domain peptide (Nixon, et al. Curr. Opin. Drug Discov. Devel. 9 (2): 261-8 (2006)), and/or a monobody (Koide, et al. Methods Mol. Biol. 352: 95-109 (2007)). Other mimetics may include, for example, a derivative of an antibody such as, for example, an Fab, Fab2, Fab′ single chain antibody, Fv, single domain antibody, mono-specific antibody, bi-specific antibody, tri-specific antibody, multi-valent antibody, chimeric antibody, canine-human chimeric antibody, canine-mouse chimeric antibody, antibody comprising a canine Fc, humanized antibody, human antibody, caninized, CDR-grafted antibody, shark antibody, nanobody, camelid antibody, microbody, and/or intrabody; and/or derivative thereof. Other binding agents are also provided herein as would be understood by one of ordinary skill in the art.
Any method known to those of ordinary skill in the art may be used to generate binding agents having specificity for (e.g., binding to) HIV. For instance, to generate and isolate monoclonal antibodies an animal such as a mouse may be administered (e.g., immunized) with one or more HIV proteins. Animals exhibiting serum reactivity to HIV expressed on activated human T lymphocytes (as determined by, for instance, flow cytometry and/or microscopy) may then be selected for generation of anti-HIV hybridoma cell lines. This may be repeated for multiple rounds. Screening may also include, for instance, affinity binding and/or functional characterization to identify the binding agent as being specific for HIV. In some embodiments, such as in the Examples herein, human beings may be screened for the expression of antibodies against HIV. In some embodiments, plasma samples of human beings infected by HIV may be screened to identify persons expressing anti-HIV antibodies, and in particular, neutralizing antibodies. Neutralizing antibody-producing cells of such persons may then be isolated, followed by the isolation and characterization of the antibodies produced thereby (e.g., as in the examples herein). A neutralizing antibody may be one that exhibits the ability to neutralize, or inhibit, infection of cells by HIV. In general, a neutralization assay typically measures the loss of infectivity of the virus through reaction of the virus with specific antibodies. Typically, a loss of infectivity is caused by interference by the bound antibody with any of the virus replication steps including but not limited to binding to target cells, entry, and/or viral release. The presence of un-neutralized virus is detected after a predetermined amount of time, e.g., one, two, three, four, five, six, seven, eight, nine, 10, 12 or 14 days, by measuring the infection of target cells using any of the systems available to those of ordinary skill in the art (e.g., a luciferase-based system). A non-limiting example of a neutralization assay may include combining a given amount of a virus or pseudovirus (see below) and different concentrations of the test or control (typically positive and negative controls assayed separately) antibody or antibodies are mixed under appropriate conditions (e.g., one (1) hour at room temperature) and then inoculated into an appropriate target cell culture (e.g., TZM-bl cells). For instance, binding agent-producing cells (e.g., B cells producing antibodies) may be assayed for the production of HIV-1 neutralizing antibodies by seeding such cells in separate plates as single cell micro-cultures on human feeder cells in the presence of Epstein-Barr Virus (EBV) (which also stimulate polyclonally memory B cells), a cocktail of growth factors (e.g., TLR9 agonist CpG-2006, IL-2 (1000 IU/ml), IL-6 (10 ng/ml), IL-21 (10 ng/ml), and anti-B cell receptor (BCR) goat antibodies (which trigger BCRs). After an appropriate time (e.g., 14 days), supernatants of such cultures may tested in a primary luciferase-based screening system using two or more representative HIV-1 viruses or pseudoviruses that productively infect such cells. The pseudoviruses may be incubated with B cell culture supernatants for an appropriate time and temperature (e.g., one (1) h at 37% (5% CO2)) before the addition of host cells (e.g., 3000 TZM-bl cells). Incubation for an appropriate time (e.g., 72 hours) may then follow, after which the supernatant may be removed and Steadylite reagent (Perkin Elmer) added (e.g., 15 μl). Luciferase activity may then determined (e.g., five minutes later) on a Synergy microplate luminometer (BioTek). Decreased luciferase activity relative to a negative control typically indicates virus neutralization. Neutralization assays such as these, suitable for analyzing binding agents of this disclosure, are known in the art (see, e.g., Montefiori, D. C. Curr. Protocol. Immunol. Chapter 12, Unit 12.11 (2005); Edmonds, et al. Virology, 408(1): 1-13 (2010); Seaman, et al. J. Virol. 84(3): 1439-1452 (2010); Pace, et al. PNAS USA, 110(33): 13540-13545 (2013)). In some embodiments, test samples may be screened for the presence of antibodies able to neutralize a panel of HIV pseudoviruses (e.g., eight (8) HIV-1 pseudoviruses from the Global Panel of HIV-1 reference strains as conducted in the examples herein (those pseudoviruses being TRO.11 (B), 246F3 (AC), BJOX2000 (CRF007_BC), CE1176 (C), CH119 (CRF07_BC), CNE55 (CRF01_AE), 25710 (C), and X1632 (see, e.g.,
In some embodiments, the binding agents described herein may be broadly neutralizing antibodies (bNabs) identified in biological samples (e.g., plasma) obtained from HIV-infected persons. As mentioned above and shown in the examples herein, such bNabs may be identified by testing plasma samples of patients chronically infected by HIV (preferably those naïve to antiretroviral therapy) for the ability to neutralize multi-Glade HIV isolates (e.g., initially using a nine or 12-member panel and then a larger panel (e.g., 57 members) of pseudoviruses)). In some embodiments, the samples may be derived from patients known to be “Elite Controllers” with viremia <50 HIV RNA copies per ml of plasma. Screening procedures such as these may result in the identification of patients that may serve as lymph node donors for the subsequent isolation and characterization of B cells producing bNabs. In carrying out such screening assays, neutralizing activity is typically compared to a negative control such as murine leukemia virus (MLV) pseudovirus.
In some embodiments, germinal center and memory IgG B cells of patients expressing neutralizing binding agents (e.g., antibodies) may be isolated and further studied. In some embodiments, the cells may be sorted separately according to IgG (e.g., IgA and IgM negative cells), CD19, and CD38 expression (germinal center B cells are CD38 positive) and interrogated for the production of HIV-1 neutralizing antibodies. For instance, highly pure IgG memory B cells and IgG germinal cells may be seeded in separate plates as single cell micro-cultures on human feeder cells in the presence of Epstein-Barr Virus (EBV) (which also stimulate polyclonally memory B cells) and a cocktail of growth factors and the like (e.g., composed TLR9 agonist CpG-2006, IL-2 (1000 IU/ml), IL-6 (10 ng/ml), IL-21 (10 ng/ml), and anti-BCR goat antibodies (B cell receptor (BCR) triggering)). Supernatants of such cultures (e.g., from day 14 cultures) may then be tested in a primary screening (e.g., using a 384-well based HIV-1 pseudoviruses neutralization assay using in parallel two strains, CE1176 and BJOX2000, representative of Glade C and CRF07, as shown in the examples herein). Neutralization assays may be carried out using any suitable host cells (e.g., TZM-bl cells (Seaman, et al. J. Virol. 84(3): 1439-52 (2010); NIH AIDS Reagent Program Catalog Number 8129)). HIV-1 pseudoviruses resulting in a significant output relative light units (RLU) (e.g., of 50-100×104 RLU) (i.e., indicating productive infection of cells) may then incubated with B cell culture supernatants for an appropriate time and temperature (e.g., one (1) h at 37% (5% CO2)) before the addition of host cells (e.g., 3000 TZM-bl cells). Incubation for an appropriate time (e.g., 72 hours) typically follows, after which the supernatant may be removed and Steadylite reagent (Perkin Elmer) added (e.g., 15 μl). Luciferase activity may then be detected (e.g., five minutes later) on a Synergy microplate luminometer (BioTek). Decreased luciferase activity indicates a lesser amount of virus being released by the cells and virus neutralization. For instance, if the base RLU for a particular pseudovirus is 50-100×104 RLU, a neutralizing antibody may be determined to decrease the RLU for that pseudovirus to 25-50×104 RLU (i.e., a 50% decrease), or less. Using such systems, supernatants capable of cross-neutralizing strains may be identified, further harvested, and tested for their ability to neutralize other pseudoviruses.
The antibodies derived from such neutralizing antibody-containing cultures may then be further characterized by determining the amino acid and nucleotide sequences of the antibody variable and complementarity determining regions (CDRs) regions. Using these techniques, the HIV-neutralizing binding agent termed “LN02” was identified as an IgG3-type fully human monoclonal antibody having the CDR, VH and VL sequences shown in
In some embodiments, the binding agents may be tested for neutralization capacity against HIV reference pseudoviruses (e.g., the above-described Global Panel of nine (9) HIV-1 reference pseudoviruses) using cells expressing or not expressing one or more types of Fc receptors (e.g., parental TZM-bl cells and TZM-bl cells expressing Fc-gamma receptor I (CD64) as in the examples; see e.g. Perez, et al. Utilization of immunoglobulin G Fc receptors by human immunodeficiency virus type 1: a specific role for antibodies against the membrane-proximal external region of gp41. J Virol 83, 7397-7410 (2009); NIH AIDS Reagent Program Catalog No. 11798). Enhanced neutralizing activity in cells expressing Fc receptors may provide antibodies a kinetic advantage for virus inhibition. This kinetic advantage could be unique to antibodies, whose epitopes are thought to be difficult to access or exposed for only a short time on intermediate conformations of the Env protein during an early stage of fusion. Fc-gamma receptors could also potentially facilitate HIV-1 neutralization is phagocytosis, thereby increasing neutralization capacity of the antibodies. To this point, HeLa cells, from which the TZM-bl cell line was constructed, are known to exhibit properties of nonprofessional phagocytes. Thus, it is possible that TZM-bl cells were converted to professional phagocytic cells by introducing Fc-gamma receptor on their surface. Any Fc-gamma-receptor-mediated antiviral effects on HIV-1 neutralizing antibodies, whether by entry inhibition or phagocytosis, might be beneficial in HIV treatment and vaccine regimens. Fc-gamma receptors are rarely expressed on CD4+ lymphocytes, but several other HIV-1-susceptible cell types express multiple Fc-gamma receptors and are involved in sexual transmission and the early establishment of long-lived viral reservoirs. In particular, macrophages are among the first infection-susceptible cells that the virus encounters after mucosal exposure, and are thought to serve as a long-lived virus reservoir in chronic infection. Macrophages, as well as certain subsets of monocytes and dendritic cells, are known to express multiple Fc-gamma receptors. It is also important to mention that Fc-gamma receptors play a role in regulating adaptive immunity and peripheral tolerance, by facilitating antigen uptake, antigen presentation, cell activation and B cell tolerance. Thus, is some embodiments, the binding agents described herein may be used in conjunction with agents that induce and/or enhance Fc receptor expression, including the introduction of nucleic acids encoding one or more Fc receptors with or in conjunction with treatment by the binding agents described herein.
The specificity of the binding agents described herein may be determined using any of the many techniques available to those of ordinary skill in the art. For instance, as shown in the examples herein, the specificity of a binding agent (e.g., IgG1 LN02 antibody), with respect to particular epitopes, may be ascertained using a panel of pseudoviruses (e.g., CAP45) that encode mutations in the HIV envelope gene. For instance, modifications can be made to HIV envelope protein (Env) to produce modified Env proteins (mEnv) and each binding agent (e.g., antibody) tested for its ability to bind to the various mEnv proteins. An exemplary HIV-1 envelope amino acid sequence that can be used is that of the CAP45 pseudovirus (GenBank Accession No μF203962; NCBI GenPept Accession No.
ABQ02701.1; SEQ ID NO.: 237) and/or the HXB2 Env sequence (SEQ ID NO.: 238 (GenBank Accession No. MF944225.1, protein_id=ATG88205.1)) as shown below (with exemplary amino acids that could be modified indicated in bold and underlined):
VTE NFNMWKNDMV DQMHEDIISL WDQSLKPCVK
RR
V
R
G
GFLGAAGST MGAASITLTV QARQLLSGIV QQQSNLLRAI
NKS
MTWI QWDREISNYS NTIYKLLEDS QNQQEQNEKD LLALDSWNNL WNWFNIT
WL
VT ENFNMWKNDM VEQMHEDIIS LWDQSLKPCV
KRR
V
R
G
GFLGAAGSTM GAASMTLTVQ
NKS
E QIWN
TTWME WDREINNYTS LIHSLIEESQ NQQEKNEQEL
WLW YIKLFIMIVG GLVGLRIVFA VLSVVNRVRQ GYSPLSFQTH
In some embodiments, a binding agent of this disclosure may comprise these binding specificities along with the neutralization characteristics described above (i.e., neutralization of HIV-1 pseudoviruses TRO.11 (Clade B), 25710 (Clade C), CE1176, BJOX (CRF07_BC), CH119 (CRF07_BC), 246-F3 (Clade AC), X1632 (Clade G), CNE55 (CRF01_AE), CD0217(Clade C) at a concentration is from 10−2 to 10° μg/ml (i.e., 10 ng/ml to 1 μg/ml), or between 100-101 μg/ml (i.e., 1-10 μg/ml), to at least about 50% (
The specificity of a binding agent may also be tested for binding to soluble trimers representing HIV proteins (e.g., soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505 as used in the examples herein). Preferred trimers (such as those used in the examples herein) are those being highly stable, homogenous and closely resembling native virus spikes when visualized by negative stain electron microscopy (EM) (Sanders, R. W. et al. A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. PLoS Pathog. 9, e1003618 (2013)). Typically, broadly neutralizing antibodies against multiple neutralizing epitopes on HIV-1 Env will be highly reactive with such trimers. Conversely, non-neutralizing antibodies (NAbs) to the CD4-binding site, CD4-induced epitopes or gp41 ectodomain would not (and did not in the example) react with the trimers, even when their epitopes were present on simpler forms of Env (e.g., gp120 monomers or dissociated gp41 subunits). The examples also included a test, which may be used in testing any of binding agents described herein, in which the MPER was also deleted to improve trimer solubility and reduce aggregate formation. The binding agents may also be tested for binding to such trimers in the presence or absence of soluble CD4 (sCD4). The examples herein describe the testing of the LN02 and PGT151 (binding to a site at the interface between gp120 and gp41 (Dingens et al. Cell Host Microbe. 2017 Jun. 14; 21(6):777-787.e4. doi: 10.1016/j.chom.2017.05.003. Epub 2017 Jun. 1)) antibodies for binding to the 426c WT SOSIP Env protein complex, measured by surface plasmon resonance (SPR)). As shown in the examples, the biotinylated IgG1 LN02 antibody was shown to bind the 426c WT SOSIP Env protein but was completely blocked from binding the Env protein that had been pre-incubated with an unlabeled LN02 antibody. In a similar experiment, the biotinylated interface binding bNab PGT151 bound tightly to 426c WT SOSIP Env protein but PGT151 bound more weakly to the LN02+426c WT SOSIP Env protein complex. The results presented in the examples therefore indicate that IgG1 LN02 antibody might recognize an epitope at the gp120/gp41 interface of HIV-1 Env. Given that PGT151 binding to 426c WT SOSIP was not completely blocked by LN02, it is possible that LN02 binds in the same region but not identical epitope compared to PGT151. Other assay systems including surface plasmon resonance may be used to test the binding agents contemplated herein. And similar tests may also be performed on any of the binding agents contemplated herein.
The term “binding affinity” and/or KD refers to the dissociation rate of a particular antibody-antigen interaction. The KD is the ratio of the rate of dissociation (“off-rate (kd)”) to the association rate (“on-rate (ka)). KD therefore equals kd/ka and is expressed as a molar concentration (M). Thus, the smaller the KD, the stronger the affinity of binding. For example, a KD of 1 mM indicates weak binding as compared to a KD of 1 nM. KD values for antibodies can be determined using methods well established in the art such as by using a Biacore® system. In some embodiments, the binding agents described herein may be compared with another binding agent with reference to the respective KD values of each. These properties may be combined with other characteristics such as neutralization capacity and/or epitope specificity in order to compare binding agents to one another. Accordingly, binding agents having a similar KD to those described herein, perhaps also sharing the neutralization capacity and epitope specificity described herein (e.g., as exhibited by LN02M), are also contemplated as part of this disclosure.
Any of the amino acid sequences of LN02M variable region and/or CDR and/or non-CDR amino acid sequences (and/or any one or more fragments and/or derivatives thereof) may be also substituted by any other amino acid as desired by one of ordinary skill in the art. For example, one of skill in the art may make conservative substitutions by replacing particular amino acids with others as shown in Table 5 below. The specific amino acid substitution selected may depend on the location of the site selected. An amino acid substitution may be said to “correspond to” where one of ordinary skill in the art could ascertain a significant amount of similarity between the amino acid sequences surrounding the amino acid being substituted. For instance, a particular amino acid sequence may correspond to another where two, three, four or more N-terminal and C-terminal amino acids surrounding the amino acid being substituted are the same or similar (e.g., as described in Table 5) in the polypeptides being compared. Conservative amino acid substitutions may involve a substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position and, in particular, does not result in, e.g., decreased HIV neutralization capacity and/or different epitope specificity.
In certain embodiments, a nucleic acid molecule encoding one or more binding agents described herein may be inserted into one or more expression vectors, as discussed below in greater detail. In such embodiments, the binding agent may be encoded by nucleotides corresponding to the amino acid sequence. The particular combinations of nucleotides (codons) that encode the various amino acids (AA) are well known in the art, as described in various references used by those skilled in the art (e.g., Lewin, B. Genes V, Oxford University Press, 1994). The nucleotide sequences encoding the amino acids of said binding agents may be ascertained with reference to Table 6, for example. Nucleic acid variants may use any combination of nucleotides that encode the binding agent.
Those of ordinary skill in the art understand that the nucleotide sequence encoding a particular amino acid sequence may be easily derived from the amino acid sequence of any of LN02M variable region and/or CDR and/or non-CDR amino acid sequences and the information presented in Table 6. For instance, it may be deduced from the amino acid sequence YGSISRHFWG (SEQ ID NO.: 1) and the information presented in Table 6 that the amino acid sequence may be encoded by the nucleotide sequence TATGGCAGCATTAGCCGCCATTTTTGGGGC (SEQ ID NO.: 34). Those of ordinary skill in the art would understand that nucleotide sequences encoding LN02M variable region and/or CDR and/or non-CDR amino acid sequences may be deduced in the same way, and such nucleotide sequences are contemplated herein. Expression vectors comprising such nucleic acid sequences are also contemplated by this disclosure. Where the binding agents are antibodies, nucleotide sequences encoding the variable regions thereof may also be isolated from the phage and/or hybridoma cells expressing the same cloned into expression vectors. Methods for producing such preparations are well-known in the art.
Nucleic acid molecules encoding one or more HIV binding agents may be contained within a viral and/or a non-viral vector. In one embodiment, a DNA vector is utilized to deliver nucleic acids encoding one or more HIV binding agents to the patient. In doing so, various strategies may be utilized to improve the efficiency of such mechanisms including, for example, the use of self-replicating viral replicons (Caley, et al. 1999. Vaccine, 17: 3124-2135; Dubensky, et al. 2000. Mol. Med. 6: 723-732; Leitner, et al. 2000. Cancer Res. 60: 51-55), codon optimization (Liu, et al. 2000. Mol. Ther., 1: 497-500; Dubensky, supra; Huang, et al. 2001. J. Virol. 75: 4947-4951), in vivo electroporation (Widera, et al. 2000. J. Immunol. 164: 4635-3640), incorporation of nucleic acids encoding co-stimulatory molecules, cytokines and/or chemokines (Xiang, et al. 1995. Immunity, 2: 129-135; Kim, et al. 1998. Eur. J. Immunol., 28: 1089-1103; Iwasaki, et al. 1997. J. Immunol. 158: 4591-3301; Sheerlinck, et al. 2001. Vaccine, 19: 2647-2656), incorporation of stimulatory motifs such as CpG (Gurunathan, supra; Leitner, supra), sequences for targeting of the endocytic or ubiquitin-processing pathways (Thomson, et al. 1998. J. Virol. 72: 2246-2252; Velders, et al. 2001. J. Immunol. 166: 5366-5373), prime-boost regimens (Gurunathan, supra; Sullivan, et al. 2000. Nature, 408: 605-609; Hanke, et al. 1998. Vaccine, 16: 439-445; Amara, et al. 2001. Science, 292: 69-74), proteasome-sensitive cleavage sites, and the use of mucosal delivery vectors such as Salmonella (Darji, et al. 1997. Cell, 91: 765-775; Woo, et al. 2001. Vaccine, 19: 2945-2954). Other methods are known in the art, some of which are described below. Various viral vectors that have been successfully utilized for introducing a nucleic acid to a host include retrovirus, adenovirus, adeno-associated virus (AAV), herpes virus, and poxvirus, among others. The vectors may be constructed using standard recombinant techniques widely available to one skilled in the art. Such techniques may be found in common molecular biology references such as Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), and PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, ca). “Non-viral” plasmid vectors may also be suitable in certain embodiments. Preferred plasmid vectors are compatible with bacterial, insect, and/or mammalian host cells. Such vectors include, for example, PCR-ii, PCR3, and pcDNA3.1 (Invitrogen, San Diego, Calif.), pBSii (Stratagene, La Jolla, Calif.), pet15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFp-n2 (Clontech, Palo Alto, Calif.), pETI (Bluebacii, Invitrogen), pDSR-alpha (PCT pub. No. WO 90/14363) and pFASTBACdual (Gibco-BRL, Grand island, NY) as well as Bluescript® plasmid derivatives (a high copy number COLe1-based phagemid, Stratagene Cloning Systems, La Jolla, Calif.), PCR cloning plasmids designed for cloning TAQ-amplified PCR products (e.g., TOPO™ TA cloning® kit, PCR2.1® plasmid derivatives, Invitrogen, Carlsbad, Calif.). Bacterial vectors may also be used. These vectors include, for example, Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille Calmette Guérin (BCG), and Streptococcus (see for example, WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796; and WO 92/21376). Many other non-viral plasmid expression vectors and systems are known in the art and may be use. Other delivery techniques may also suffice including, for example, DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO4 precipitation, gene gun techniques, electroporation, and colloidal dispersion systems. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system is a liposome, which are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, R., et al., 1981, Trends Biochem. Sci., 6: 77). The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
A cultured cell comprising the vector is also provided. The cultured cell may be a cultured cell transfected with the vector or a progeny of the cell, wherein the cell expresses the immunogenic polypeptide. Suitable cell lines are known to those of skill in the art and are commercially available, for example, through the American Type Culture Collection (ATCC). The transfected cells can be used in a method of producing an immunogenic polypeptide. The method comprises culturing a cell comprising the vector under conditions that allow expression of the immunogenic polypeptide, optionally under the control of an expression sequence. The immunogenic polypeptide can be isolated from the cell or the culture medium using standard protein purification methods. In some embodiments, the binding agents described herein may be conjugated to active agents to target and inhibit the function of and/or eliminate cell populations expressing HIV polypeptides and/or harboring HIV (and/or another antigen in the case of binding agents with multiple specificities). For instance, CD4+ T-cell populations containing replication competent HIV may be targeted and eliminated using binding agent/drug conjugates (e.g., antibody-drug conjugates (ADC)). Mono- and/or bi-specific candidate binding agents may be conjugated with one or more types of drugs (e.g., drugs damaging DNA, targeting microtubules). The binding agents described herein and/or derivatives thereof may also be adjoined to and/or conjugated to functional agents for in vitro and/or in vivo use. For instance, the binding agent may be adjoined to and/or conjugated to functional moieties such as cytotoxic drugs or toxins, and/or active fragments thereof such as diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, among others. Suitable functional moieties may also include radiochemicals. Binding agents, such as antibodies, may be adjoined to and/or conjugated to the one or more functional agents using standard techniques in the art.
In some embodiments, this disclosure provides binding agents with multiple specificities such that epitopes bound by LN02M binding agent and at least one other secondary antigen (e.g., a cell surface protein) may be bound by a single binding agent. In some embodiments, the secondary antigen may be one expressed by cells infected by an infectious agent. For instance, an exemplary secondary antigen may be HIV Env antigen other than gp41. Such binding agents may bind the secondary antigen and/or may serve to neutralize the infectious agent as may be determined using the assays described herein. Combinations of binding agents, such as one or more described herein with another available to those of ordinary skill in the art, are also contemplated herein. For instance, in some embodiments, the combinations may be identified to provide statistically significant differences from results (e.g., neutralization assays) obtained using only one or more of the binding agents and not others. In some embodiments, combinations exhibit additive and/or, preferably synergistic, neutralization of HIV, for example. In some embodiments, the combination may comprise a first binding agent having the characteristics of an LN02M binding agent (i.e., comprising a LN02M variable region and/or CDR and/or non-CDR amino acid sequences), and/or derivatives thereof, and any one or more of the antibodies described in any one or more of U.S. Pat. Nos. 5,087,557; 5,298,419; 5,459,060; 5,693,752; 5,731,189; 5,753,503; 5,756,674; 5,777,074; 5,804,440; 5,831,034; 6,008,044; 7,774,88762; U.S. Pat. Publications 2003/0118985A1, 2007/0292390A1, or 2014/0205612A1; WO 2002/032452A1 (e.g., binding the gp41 epitopes ELDKWA, ELEKWA, ELNKWA, ELDEWA); EP0335134B1 US 176077 (e.g, a humanized version of the mouse mAbs described therein); DE3932461A1 (mAb against the epitope Arg-Ile-Leu-Ala-Val-Glu-Arg-Leu-Lys-Try-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly-Cys-Ser); Evans, et al. J. Immunol. 140(3): 941-3 (1988); Gorney, et al. Proc. Natl. Acad. Sci. USA, 86: 1624-28 (1989); Teeuwsen, et al. (1990) AIDS Res. Hum. Retroviruses 6, 381-392; Earl, et al. J. Virol. 71(4): 2674-2684 (1997); Jiang, et al. J. Virol. 72(12): 10213-17 (1998); Zwick, et al. J. Virol. 75(22): 10892-10905 (2001); Eckert et al. PNAS USA, 98(20): 11187-11192 (2001); Louis, et al. J. Biol. Chem. 278(22): 20278-20285 (2003); and/or Pietzsch, et al. J. Virol. 84(10): 5032-42 (2010); all of which are incorporated herein in their entirety. For instance, any of the binding agents described herein may be combined with (i.e., as a single composition, and/or used in conjunction with) one or more the antibodies commonly known as 2F5, 4E10 and/or Z13e1, and/or derivatives thereof, among others. The binding agents of such compositions may be different entities such as two or more different monoclonal antibodies or derivatives thereof, or may be found on the same entity such as a bi-functional antibody (a single antibody or derivative thereof comprising multiple binding specificities). Such combinations as described herein may also be combined with one or more other agents that may affect immune cell function such as antibodies against CTLA-4, and the like. One of ordinary skill in the art would recognize that many such combinations may be suitable for use as described herein.
As mentioned above, the HIV binding agents described herein may be used to treat and/or prevent and/or ameliorate the symptoms of infection by HIV. As is well-known in the art, HIV isolates are now classified into discrete genetic subtypes. HIV-1 is known to comprise at least ten subtypes (A1, A2, A3, A4, B, C, D, E, F1, F2, G, H, J and K) (Taylor et al, NEJM, 359(18):1965-1966 (2008)). HIV-2 is known to include at least five subtypes (A, B, C, D, and E). Subtype B has been associated with the HIV epidemic in homosexual men and intravenous drug users worldwide. Most HIV-1 immunogens, laboratory adapted isolates, reagents and mapped epitopes belong to subtype B. In sub-Saharan Africa, India and China, areas where the incidence of new HIV infections is high, HIV-1 subtype B accounts for only a small minority of infections, and subtype HIV-1 C appears to be the most common infecting subtype. Any of these types of isolates may be addressed using the binding agents described herein. One or more binding agents may also be administered with or in conjunction with one or more agents used to prevent, treat and/or ameliorate HIV such as for example, a protease inhibitor, an HIV entry inhibitor, a reverse transcriptase inhibitor, and/or an anti-retroviral nucleoside analog. Suitable compounds include, for example, Agenerase (amprenavir), Combivir (Retrovir/Epivir), Crixivan (indinavir), Emtriva (emtricitabine), Epivir (3tc/lamivudine), Epzicom, Fortovase/Invirase (saquinavir), Fuzeon (enfuvirtide), Hivid (ddc/zalcitabine), Kaletra (lopinavir), Lexiva (Fosamprenavir), Norvir (ritonavir), Rescriptor (delavirdine), Retrovir/AZT (zidovudine), Reyatax (atazanavir, BMS-232632), Sustiva (efavirenz), Trizivir (abacavir/zidovudine/lamivudine), Truvada (Emtricitabine/Tenofovir DF), Videx (ddl/didanosine), Videx EC (ddl, didanosine), Viracept (nevirapine), Viread (tenofovir disoproxil fumarate), Zerit (d4T/stavudine), and Ziagen (abacavir) may be utilized. Other suitable agents are known to those of skill in the art and may be suitable for use as described herein. Such agents may either be used prior to, during, or after administration of the binding agents and/or use of the methods described herein.
The skilled artisan has many suitable techniques for using the binding agents (e.g., antibodies) described herein to identify biological samples containing proteins that bind thereto. For instance, antibodies may be utilized to isolate HIV or cells containing HIV and/or expressing HIV antigens using, for example, immunoprecipitation or other capture-type assay. This well-known technique is performed by attaching the antibody to a solid support or chromatographic material (e.g., a bead coated with Protein A, Protein G and/or Protein L). The bound antibody is then introduced into a solution either containing or believed to contain HIV antigens (e.g., an HIV-infected cell). The HIV antigen(s) may then bind to the antibody and non-binding materials are washed away under conditions in which the HIV antigen(s) remains bound to the antibody. The bound protein may then be separated from the antibody and analyzed as desired. Similar methods for isolating a protein using an antibody are well-known in the art. The binding agents (e.g., antibodies) may also be utilized to detect HIV or HIV antigens within a biological sample. For instance, the antibodies may be used in assays such as, for example, flow cytometric analysis, ELISA, immunoblotting (e.g., western blot), in situ detection, immunocytochemistry, and/or immunohistochemistry. Methods of carrying out such assays are well-known in the art. In some embodiments, the binding agents may be adjoined to and/or conjugated to one or more detectable labels. For instance, suitable detectable labels may include, for instance, fluorosceins (e.g., DyLight, Cy3, Cy5, FITC, HiLyte Fluor 555, HiLyte Fluor 647; 5-carboxy-2,7-d ichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 6-JOE; 6-carboxyfluorescein (6-FAM); FITC; 6-carboxy-1,4-dichloro-2′,7′-dichlorofluorescein (TET); 6-carboxy-1,4-dichloro-2′,4′, 5′, 7′-tetra-chlorofluorescein (HEX); 6-carboxy-4′,5′-dichloro-2′, 7′-dimethoxyfluorescein (JOE); Alexa fluors (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY fluorophores (e.g., 492/515, 493/503, 500/510, 505/515, 530/550, 542/563, 558/568, 564/570, 576/589, 581/591, 630/650-X, 650/665-X, 665/676, FL, FL ATP, FI-Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-X SE)), rhodamines (e.g., 110, 123, B, B 200, BB, BG, B extra, 5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-Carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Phallicidine, Phalloidine, Red, Rhod-2, ROX (6-carboxy-X-rhodamine), 5-ROX (carboxy-X-rhodamine), Sulphorhodamine B can C, Sulphorhodamine G Extra, TAMRA (6-carboxytetramethylrhodamine), Tetramethylrhodamine (TRITC), WT), Texas Red, and/or Texas Red-X. Other detectable labels known in the art may also be suitable for use. Binding agents, such as antibodies, may be adjoined to and/or conjugated to the one or more detectable labels using standard techniques in the art.
The binding agents described herein may be also be used to determine the presence of a disease state in a patient, to predict prognosis, or to determine the effectiveness of a chemotherapeutic or other treatment regimen. Expression profile assays, performed as described herein or as is otherwise known in the art, may be used to determine the relative level of expression of HIV in a cell, for instance. The level of expression may then be correlated with base (e.g., control) levels to determine whether a particular disease is present within the patient, the patient's prognosis, or whether a particular treatment regimen is effective. For example, if the patient is being treated with a particular anti-infective regimen, an increased or decreased level of expression of HIV in the patient's tissues (e.g., in plasma) may indicate the regimen is worsening or improving the load of HIV in that host. The increase or decrease in expression may indicate the regimen is having or not having the desired effect and another therapeutic modality may therefore be selected.
It is also possible to use the binding agents described herein as reagents in drug screening assays to test, for example, new drug candidates. The reagents may be used to ascertain the effect of a drug candidate on the expression of the immunogenic target in a cell line, or a cell or tissue of a patient. The expression profiling technique may be combined with high throughput screening techniques to allow rapid identification of useful compounds and monitor the effectiveness of treatment with a drug candidate (see, for example, Zlokarnik, et al., Science 279, 84-8 (1998)). Drug candidates may be chemical compounds, nucleic acids, proteins, antibodies, or derivatives therefrom, whether naturally occurring or synthetically derived. Drug candidates thus identified may be utilized, among other uses, as pharmaceutical compositions for administration to patients or for use in further screening assays.
In some embodiments, the binding agents are in purified form. A “purified” binding agent (e.g., antibody) may be one that is separated from at least about 50% of the proteins and/or other components with which it is initially found (e.g., as part of a hybridoma supernatant or ascites preparation in the case of a monoclonal antibody). A purified binding agent (e.g., antibody) may be one that is separated from at least about 50%, 60%, 75%, 90%, or 95% of the proteins and/or other components with which it is initially found.
The binding agents (e.g., polypeptides, antibodies) and nucleic acids described herein may also be combined with one or more pharmaceutically acceptable carriers prior to administration to a host. A pharmaceutically acceptable carrier is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. Suitable pharmaceutical carriers and their formulations are described in, for example, Remington's: The Science and Practice of Pharmacy, 21St Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution. The pH of the solution is generally from about 5 to about 8 or from about 7 to about 7.5. Other carriers include sustained-release preparations such as semipermeable matrices of solid hydrophobic polymers containing polypeptides or fragments thereof. Matrices may be in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Carriers are those suitable for administration of polypeptides and/or fragments thereof to humans or other subjects. Pharmaceutical compositions may also include carriers, thickeners, diluents, buffers, preservatives, surface active agents, adjuvants, immunostimulants, in addition to the immunogenic polypeptide. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents and anesthetics. The pharmaceutical composition may be administered orally, parentally, by inhalation spray, rectally, intranodally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of a nucleic acid, polypeptide, or peptide as a pharmaceutical composition. A “pharmaceutical composition” is a composition comprising a therapeutically effective amount of a nucleic acid or polypeptide. The terms “effective amount” and “therapeutically effective amount” each refer to the amount of a binding agent, nucleic acid or the like used to observe the desired therapeutic effect (e.g., eliminating HIV).
Methods for treating one or more disease conditions (e.g., HIV or cancer) in a mammalian host comprising administering to the mammal at least one or more effective doses of one or more binding agents (and/or derivative(s) thereof) described herein are also provided. In some embodiments, the binding agent is a monoclonal antibody or fragment or derivative thereof comprising one or more of LN02M variable region and/or CDR and/or non-CDR amino acid sequences (i.e., comprising LN02M variable region and/or CDR and/or non-CDR amino acid sequences). The one or more binding agents may be administered in a dosage amount of about 1 to about 50 mg/kg, about 1 to about 30 mg/kg, or about 5 to about 30 mg/kg (e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 mg/kg). In certain embodiments, the one or more binding agents may be administered to the mammal (e.g., intradermally, intravenously, orally, rectally) at about 10 mg/kg one or more times. When multiple doses are administered, the doses may comprise about the same or different amount of binding agent in each dose. The doses may also be separated in time from one another by the same or different intervals. For instance, the doses may be separated by about any of 6, 12, 24, 36, 48, 60, 72, 84, or 96 hours, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 12 months, 1.5 years, 2 years, 3 years, 4 years, 5 years, or any time period before, after, and/or between any of these time periods. In some embodiments, the binding agents may be administered in conjunction with other agents (e.g., anti-infective agents and/or chemotherapeutic agent). Such other agents may be administered about simultaneously with the binding agents, or at a different time and/or frequency. Other embodiments of such methods may also be appropriate as could be readily determined by one of ordinary skill in the art.
To assist the skilled artisan in using the binding agents such as antibodies described herein, the same may be provided in kit format. A kit including one or more of such binding agents and optionally other components necessary for using the same to detect cells expressing HIV is also provided. The binding agents of the kit may be provided in any suitable form, including frozen, lyophilized, or in a pharmaceutically acceptable buffer such as TBS or PBS. The kit may also include other reagents required for utilization of the binding agents in vitro or in vivo such as buffers (e.g., TBS, PBS), blocking agents (solutions including nonfat dry milk, normal sera, Tween-20 Detergent, BSA, or casein), and/or detection reagents (e.g., goat anti-mouse IgG biotin, streptavidin-HRP conjugates, allophycocyanin, B-phycoerythrin, R-phycoerythrin, peroxidase, detectable labels, and other labels and/or staining kits (e.g., ABC Staining Kit, Pierce)). The kits may also include other reagents and/or instructions for using the antibodies in commonly utilized assays described above such as, for example, flow cytometric analysis, ELISA, immunoblotting (e.g., western blot), in situ detection, immunocytochemistry, and/or immunohistochemistry. In one embodiment, the kit provides a binding agent in purified form. In another embodiment, the binding agent may be provided in biotinylated form either alone or along with an avidin-conjugated detection reagent (e.g., antibody). In another embodiment, the kit includes a binding agents comprising one or more detectable labels that may be used to directly detect HIV. Buffers and the like required for using any of these systems are well-known in the art and/or may be prepared by the end-user or provided as a component of the kit. The kit may also include a solid support containing positive- and negative-control protein and/or tissue samples. For example, kits for performing spotting or western blot-type assays may include control cell or tissue lysates for use in SDS-PAGE or nylon or other membranes containing pre-fixed control samples with additional space for experimental samples. Kits for visualization of HIV in cells on slides may include pre-formatted slides containing control cell or tissue samples with additional space for experimental samples. Other embodiments of kits are also contemplated herein as would be understood by those of ordinary skill in the art.
Thus, this disclosure provides binding agents such as the LN02 antibody with specificity for HIV (e.g., and/or an antigen thereof). In some embodiments, the binding agent is a polypeptide comprising at least one amino acid sequence selected from the group consisting of one or more LN02M variable region and/or CDR and/or non-CDR amino acid sequences. In some embodiments, the binding agent is a polypeptide comprising one or more combinations of LN02M variable region and/or CDR and/or non-CDR amino acid sequences. In some embodiments, the binding agent is an antibody. In some embodiments, the binding agent is a polypeptide such as an antibody comprising heavy and/or light chain CDRs and/or additional amino acid sequence shown for any of the binding agents (e.g., antibodies or derivatives thereof) of
In some embodiments, the binding agents have specificity for an epitope comprising amino acid residues in the proximity of the gp120/gp41 interface of HIV-1 Env (corresponding residues underlined in SEQ ID NO. 237). In some embodiments, a binding agent of this disclosure may comprise any one or more of these binding specificities along with the neutralization characteristics described above but not of a control virus at a concentration is from 102−10° ug/ml, or between 100-101 ug/ml, to at least about 50%, and/or the ability to the neutralize HIV-1 pseudoviruses at an IC50 or IC80 of less than 25.
In some embodiments, the binding agent is derived from or related to (e.g., by sequence or derivation) a human antibody, human IgG, human IgG1, human IgG2, human IgG2a, human IgG2b, human IgG3, human IgG4, human IgM, human IgA, human IgA1, human IgA2, human IgD, human IgE, canine antibody, canine IgGA, canine IgGB, canine IgGC, canine IgGD, chicken antibody, chicken IgA, chicken IgD, chicken IgE, chicken IgG, chicken IgM, chicken IgY, goat antibody, goat IgG, mouse antibody, mouse IgG, pig antibody, and/or rat antibody, and/or a derivative thereof. In some embodiments, the derivative may be selected from the group consisting of an Fab, Fab2, Fab′ single chain antibody, Fv, single chain, mono-specific antibody, bispecific antibody, trimeric antibody, multi-specific antibody, multivalent antibody, chimeric antibody, canine-human chimeric antibody, canine-mouse chimeric antibody, antibody comprising a canine Fc, humanized antibody, human antibody, caninized antibody, CDR-grafted antibody, shark antibody, nanobody, and/or camelid antibody. In some embodiments, the binding agent comprises at least a least a first and second specificity, the first being against HIV gp41 and the second being against a different antigen (e.g., an antigen of an infectious agent such as HIV (e.g., env) and/or a tumor antigen). In some embodiments, the binding agent and/or derivative thereof may comprise a detectable label fixably attached thereto. In some embodiments, the binding agent of any one and/or derivative thereof comprises an effector moiety (e.g., a cytotoxic drug, toxin, diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, and radiochemical) fixably attached thereto. In some embodiments, polynucleotides encoding one or more binding agents are also provided (e.g., as an expression vector). Host cells comprising and/or expressing the polypeptide products of such polynucleotides are also provided. In some embodiments, compositions comprising at least one binding agent or derivative; at least one isolated polynucleotide; at least one expression vector; and/or, at least one host cell; or a combination thereof; and, a pharmaceutically acceptable carrier are also provided.
This disclosure also provides methods for detecting HIV on a cell, the method comprising contacting a test biological sample with a binding agent or derivative described herein and detecting the binding agent bound to the biological sample or components thereof. Such methods may be an in vivo method or an in vitro method. In some embodiments, the method may comprise comparing the amount of binding to the test biological sample or components thereof to the amount of binding to a control biological sample or components thereof, wherein increased binding to the test biological sample or components thereof relative to the control biological sample or components thereof indicates the presence of a cell expressing HIV polypeptides in the test biological sample (e.g., mammalian blood). In some embodiments, a kit for detecting the expression of HIV in or on a cell, the kit comprising a binding agent or derivative thereof and instructions for use. In some embodiments, the binding agent and/or derivative thereof is in lyophilized form. In some embodiments, this disclosure provides methods for treating, preventing and/or ameliorating an infectious disease, cancer and/or autoimmunity in a mammal comprising administering to the mammal at least one effective dose of a pharmaceutical composition comprising a binding agent or derivative thereof. In some embodiments, the infectious disease is human immunodeficiency virus (HIV). In some embodiments, multiple doses are administered to the animal. In some embodiments, the binding agent and/or derivative thereof may be administered in a dosage amount of about 1 to 50 mg/kg.
In some embodiments, this disclosure provides a binding agent(s) comprising a variable region shown in
In some embodiments, this disclosure provides an isolated polynucleotide encoding any such binding agent(s), expression vectors comprising the same, and/or a host cell comprising the same. In some embodiments, this disclosure provides a composition comprising at least one such binding agent and/or or derivative thereof, at least one isolated polynucleotide encoding the same; at least one expression vector encoding the same, and/or, at least one host cell capable of producing the same (e.g., comprising at least one such polynucleotide and/or expression vector), and/or a combination thereof; and, a pharmaceutically acceptable carrier. In some embodiments, this disclosure also provides methods for manufacturing a binding agent and/or derivative thereof. In some embodiments, such methods for manufacturing comprising expressing one or more polynucleotides encoding a binding agent and/or derivative thereof of this disclosure in a host cell and purifying (e.g., to 90%, 95%, 99%, or 100% purity as may be determined by those of ordinary skill in the art using standard techniques) the same from the host cell, cell culture supernatant thereof, or the like, using standard techniques.
In some embodiments, this disclosure provides methods for detecting HIV on a cell, the method comprising contacting a test biological sample with a binding agent or derivative of this disclosure, and detecting the binding agent bound to the biological sample or components thereof. In some embodiments, such methods comprise comparing the amount of binding to the test biological sample or components thereof to the amount of binding to a control biological sample or components thereof, wherein increased binding to the test biological sample or components thereof relative to the control biological sample or components thereof indicates the presence of a cell expressing HIV in the test biological sample. In some embodiments, the test biological sample comprises, is, or is derived from mammalian blood or a component thereof. In some embodiments, the method is an in vivo method or the method is an in vitro method. In some embodiments, this disclosure provides methods for treating, preventing and/or ameliorating HIV infection and/or AIDS in a mammal comprising administering to the mammal at least one effective dose of a pharmaceutical composition comprising a binding agent and/or derivative thereof of this disclosure. In some embodiments, multiple doses of such a pharmaceutical composition are administered to the animal. In some embodiments, the binding agent and/or derivative thereof can be administered in a dosage amount of about 1 to 50 mg/kg. In some embodiments, this disclosure provides kits for detecting the expression of HIV in or on a cell, the kit comprising a binding agent and/or derivative thereof of this disclosure and optionally instructions for use. In some such embodiments, the binding agent, antibody, or derivative can be in lyophilized form.
The terms “about”, “approximately”, and the like, when preceding a list of numerical values or range, refer to each individual value in the list or range independently as if each individual value in the list or range was immediately preceded by that term. The terms mean that the values to which the same refer are exactly, close to, or similar thereto.
As used herein, a subject or a host is meant to be an individual. The subject can include domesticated animals, such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratory animals (e.g., mice, rabbits, rats, guinea pigs) and birds. In one aspect, the subject is a mammal such as a primate or a human.
Optional or optionally means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the phrase optionally the composition can comprise a combination means that the composition may comprise a combination of different molecules or may not include a combination such that the description includes both the combination and the absence of the combination (i.e., individual members of the combination).
Ranges may be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about or approximately, it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Ranges (e.g., 90-100%) are meant to include the range per se as well as each independent value within the range as if each value was individually listed.
The term “combined” or “in combination” or “in conjunction” may refer to a physical combination of agents that are administered together or the use of two or more agents in a regimen (e.g., administered separately, physically and/or in time) for treating, preventing and/or ameliorating a particular disease.
When the terms treat, prevent, and/or ameliorate or derivatives thereof are used herein in connection with a given treatment for a given condition (e.g., preventing cancer infection by HIV), it is meant to convey that the treated patient either does not develop a clinically observable level of the condition at all, or develops it more slowly and/or to a lesser degree than he/she would have absent the treatment. These terms are not limited solely to a situation in which the patient experiences no aspect of the condition whatsoever. For example, a treatment will be said to have prevented the condition if it is given during exposure of a patient to a stimulus that would have been expected to produce a given manifestation of the condition, and results in the patient's experiencing fewer and/or milder symptoms of the condition than otherwise expected. For instance, a treatment can “prevent” infection by resulting in the patient's displaying only mild overt symptoms of the infection; it does not imply that there must have been no penetration of any cell by the infecting microorganism.
Similarly, reduce, reducing, and reduction as used herein in connection with prevention, treatment and/or amelioration of a given condition by a particular treatment typically refers to a subject developing an infection more slowly or to a lesser degree as compared to a control or basal level of developing an infection in the absence of a treatment (e.g., administration of one or more HIV binding agents). A reduction in the risk of infection may result in the patient's displaying only mild overt symptoms of the infection or delayed symptoms of infection; it does not imply that there must have been no penetration of any cell by the infecting microorganism.
All references cited within this disclosure are hereby incorporated by reference in their entirety. Certain embodiments are further described in the following examples. These embodiments are provided as examples only and are not intended to limit the scope of the claims in any way.
Selection of HIV-1 lymph node donors for the isolation of broadly neutralizing antibodies. As described in more detail elsewhere, in order to isolate the broadly neutralizing LN02 antibodies capable to broadly neutralize multi-Glade HIV-1 isolates in 107 plasma samples from chronically infected patients naïve to antiretroviral therapy were screened for the presence of high titers of antibodies able to neutralize a panel of nine (9) HIV-1 pseudoviruses from the Global Panel of HIV-1 reference strains (DeCamp, A. et al. Global panel of HIV-1 Env reference strains for standardized assessments of vaccine-elicited neutralizing antibodies. J Virol 88, 2489-2507 (2014)). This analysis resulted in the identification of eight (8) patients (
The LN02 antibody was characterized by determining the amino acid and nucleotide sequences of its variable regions and the complementarity determining regions (CDRs) ascertained. Accordingly, the binding agent termed “LN02” is an IgG1-type fully human monoclonal antibody having the CDR, VH and VL sequences shown in
Modified LN02 (LN02M) antibodies comprising modified CDR and non-CDR amino acid sequences were also produced using recombinant techniques. In order to identify LN02M broadly neutralizing antibodies with improved viral neutralization properties, a panel of single or multiple amino acid substitutions in LN02 were generated by site directed mutagenesis in expression vectors encoding the heavy or light chain sequences of the LN02 antibody. LN02M antibodies were produced by transient transfection of CHO cells with the wild type vector of the light chain co-transfected with one of the mutant vector for the heavy chain (Table 1,
Table 1 describes the neutralization activity of antibodies comprising the LN02M variable heavy chain amino acid sequences of SEQ ID NOS. 3-92 (identified in Table 1 and
Surprisingly, several of the LN02M antibodies showed higher neutralizing activity than LN02; these include, for instance, the LN02M variable heavy chain regions MH01 (1.59), MH16 (1.69), MH22 (1.18), MH26 (1.40), MH30 (3.37), MH32 (1.32), MH35 (1.91), MH36 (1.37), MH37 (1.75), MH43 (1.90), MH44 (1.38), MH48 (2.12), MH49 (1.71), MH50 (2.74), MH51 (2.46), MH53 (1.45), MH59 (1.31), MH61 (1.43), MH64 (1.52), MH68 (1.12), MH73 (1.83), MH84 (1.16), MH89 (2.26), MH91 (1.36), MH92 (1.45), MH106 (1.16), MH107 (2.19), MH108 (1.91), MH111 (3.34), MH112 (2.77), MH115 (1.41), MH119 (1.32), MH120 (1.55), MH124 (1.67), MH 131 (1.55), MH135 (1.60), MH136 (1.84), MH138 (1.20), and MH146 (1.65); as well as the LN02M variable light chain regions ML01 (1.29), ML02 (1.93), ML05 (1.45), ML08 (2.31), ML10 (1.51), ML11 (1.25), ML12 (3.90), ML31 (5.74), ML32 (1.38), ML44 (1.57), ML49 (1.40), ML51 (1.10), ML52 (1.36), ML60 (1.17), ML71 (1.38), ML73 (1.20), ML74 (1.10), ML79 (1.46), ML84 (1.59), ML85 (9.94), and ML94 (6.42). Of note, LN02H antibodies comprising mutations LN02 MH30, LN02 MH111, LN02 ML12, LN02 ML31, LN02 ML85, LN02 ML92 and LN02 ML94 in Table 1 and 2 all demonstrate greater than 3-fold improved neutralization potency against the BaL virus relative the LN02 wild type control.
Neutralization of LN02 bNab and LN02 mutant variants against a global panel of eight pseudo-typed HIV-1 viral strains. A preliminary evaluation of the neutralization breadth of a select panel of LN02 bNabs variants with mutations in the heavy and/or light chain of LN02 was performed using a panel of eight pseudo-typed HIV-1 viruses. A summary of the 80% inhibitory concentration (IC80) for each of the LN02 mutants (MH for heavy chain mutations, ML for light chain mutants and MX for mutations in both the heavy and light chain) with each of the eight pseudo-typed viruses (TRO.11, 25710, CD1176, BJOX, CH119, 246-F3, X1632, and CNE55) is shown in
While certain embodiments have been described in terms of the preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations that come within the scope of the following claims.
This application claims priority to U.S. Ser. No. 62/874,042 filed Jul. 15, 2019 and U.S. Ser. No. 62/874,057 filed on Jul. 15, 2019, each of which being incorporated into this disclosure in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2020/056668 | 7/15/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62874042 | Jul 2019 | US | |
| 62874057 | Jul 2019 | US |
