Treatment and functional cure of HIV infection by monoclonal antibody-mediated competitive HIV entry inhibition against CD4

Abstract

The present invention relates to compositions and methods for preventing, treating, and / or functionally curing HIV infection. Aspects of the invention relate to monoclonal antibodies against CD4, compositions thereof, and methods of using such compositions to prevent, treat, and functionally cure HIV infection.

Description

TECHNICAL FIELD

[0001] The present invention relates to monoclonal antibodies against CD4 mediating competitive HIV entry inhibition, compositions thereof, and methods of using the compositions to treat and functionally cure HIV infection. BACKGROUND

[0002] Acquired Immune Deficiency Syndrome (AIDS) is a disease of the human immune system caused by the Human Immunodeficiency Virus (HIV) (http: / / en.wikipedia.org / wiki / HIV / AIDS). Genetic studies indicate that HIV originated in western central Africa in the late 1800s or early 1900s. AIDS was first recognized in 1981 by the U.S. Centers for Disease Control and Prevention, and its cause, HIV, was identified in the early 1980s. Since the beginning of the epidemic, more than 7 million people have been infected with HIV, and 3.5 million have died of AIDS. Worldwide, 34.0 million people were living with HIV at the end of 2011 (http: / / www.who.int / gho / hiv / en / ).

[0003] HIV infection progressively reduces the effectiveness of the immune system, and makes individuals susceptible to opportunistic infections and neoplasms. HIV can be transmitted through direct contact with mucous membranes or blood containing HIV-containing body fluids (e.g., blood, semen, vaginal fluid, pre-ejaculatory fluid, and breast milk). This transmission can involve anal, vaginal, or oral sex, blood transfusion, contaminated needles, mother-to-child transmission during pregnancy, childbirth, or breastfeeding, or other exposure to these fluids.

[0004] For 30 years, scientists have thought that AIDS is caused by "virus-making" CD4 T cells, not silent T cells. But these "virus-making" infected cells are not enough to explain the massive depletion of T cells in patients developing AIDS. Greene and colleagues point out that 95% of the silent lymphoid CD4 T cells die by pyroptosis, a highly inflammatory form of programmed cell death that involves the release of proinflammatory cytokines, triggered by failed viral infection (Doitsh, G. et al. 2014). These cells have cytoplasmic viral DNA, but unlike T cells that become viral-replicating units, these "HIV-infected" silent T cells self-destruct by a highly inflammatory form of programmed cell death (pyroptosis) that involves the release of proinflammatory cytokines. A cell protein, interferon-gamma-inducible protein 16 (IFI16), can recognize viral DNA and triggers a cascade of reactions in T cells, including activation of caspase-1 enzyme that mediates pyroptosis, and causes cell swelling, cell membrane permeability, and leakage of cytoplasmic contents. The self-destruction of silent T cells does not help to kill the virus. This process ultimately leads to the pathogenic mechanism of HIV to shift to drive the course of disease to AIDS.

[0005] Current treatment of HIV infection to delay the onset of AIDS is by highly active antiretroviral therapy or HAART to prevent viral replication. The current preferred HAART includes combination therapy (or "cocktail") consisting of at least three drugs belonging to at least two classes of antiretroviral drugs (Combination antiretroviral therapy, cART). A typical treatment regimen includes two nucleoside analog reverse transcriptase inhibitors (NARTIs or NRTIs) plus one protease inhibitor or non-nucleoside reverse transcriptase inhibitor (NNRTI).

[0006] In developed countries, doctors assess viral load, CD4 T cell number, rate of CD4-positive cell reduction, and patient readiness when deciding when to recommend starting HAART treatment. Generally, treatment is recommended for asymptomatic patients whose CD4 T cell number has dropped to 200-250 cells per milliliter of blood. However, starting treatment earlier (at CD4 levels of 350 cells per microliter) can significantly reduce the risk of AIDS and death.

[0007] Without treatment, the median net survival after HIV infection is 9 to 11 years, depending on the HIV subtype; and the average survival after AIDS diagnosis ranges from 6 to 19 months. In areas where HAART is widely used, the mortality of the disease has been reduced by 80%, which in turn increases the life expectancy of newly diagnosed HIV-infected patients to about 20 years.

[0008] Standard goals of HAART include improvement in the quality of life of the patient, reduction of complications, and reduction of HIV viremia to below the limit of detection.

[0009] However, HAART therapy has several problems. First, it does not cure HIV-infected patients, and once treatment is stopped, most "HAART-resistant" HIV rebounds to high blood levels. Second, HAART has unpleasant side effects including fatigue, weakness, diarrhea, headache, nausea, and vomiting. In the long term, HIV-infected patients can experience neurocognitive disorders, osteoporosis, neuropathy, cancer, kidney disease, and cardiovascular disease. Although newer antiretroviral drugs have fewer side effects than older drugs, life-long use still results in adverse outcomes. Irregular medication means HIV rebound, drug resistance, and disease progression. Third, HAART performance is far from ideal for more than 50% of patients because of drug intolerance, past ineffective antiviral therapy, and infection with drug-resistant strains of HIV.

[0010] An increasing number of researchers are studying how to cure HIV infection, or at least achieve long-term or permanent remission without antiretroviral drug therapy.

[0011] Two therapeutic strategies, eradication (i.e., eradication) and functional cure, are current research for HIV infection (as shown in Table 1). The goal of the eradication cure method is to eliminate all HIV-infected cells, completely clear HIV from the body, and is defined as reducing viral load to less than 1 copy per milliliter of blood without antiretroviral therapy. The goal of the functional cure is the remission state and long-term control of HIV, including low viral load without antiretroviral therapy, which reduces viral load to less than 50 copies per milliliter of blood for a permanent or extended period.

[0012] The only current example of a "cure" is a case study from a man infected with HIV, nicknamed the "Berlin patient," who developed acute myeloid leukemia and received a bone marrow transplant from a donor with a mutation or different form of the CCR5 gene. Forty-five months after stopping treatment, doctors could no longer detect HIV in his body. However, the strategy of bone marrow transplant with a donor having a mutation in CCR5 is not a viable HIV cure method, as it brings toxicity and complexity to the treatment. Natural cases of "functional cure" can be found in elite controllers, which are HIV-infected individuals whose immune system naturally controls the virus without the use of antiretroviral drugs. These individuals successfully maintain stable CD4 (white blood cell) cell counts, low or undetectable viral loads, and significantly lower amounts of "latent HIV" in their cells.

[0013] In fact, the main obstacle to a cure is the presence of a "latent HIV reservoir" that hides in the cells of the immune system (such as memory cells) with a long life during antiretroviral drug therapy, which acts on active viral infections rather than latent HIV by blocking replication. However, if this antiretroviral drug therapy is stopped, latent HIV can be activated to renew the process of HIV infection.

[0014] Current strategies against these "problematic" latent HIV reservoirs include efforts to deplete the latent reservoir through activation of viral expression under HAART therapy to eliminate infected cells, leaving only uninfected cells. One group of activators is histone deacetylase (HDAC) inhibitors (see Table 2). HDAC inhibitors are currently used as mood stabilizers, antiepileptic drugs, and anticancer treatments. The long-term effects of HDAC inhibitors are to increase the risk of reactivation of malignant tumors and / or oncogenes, which remains a major problem. This strategy is viable if active viral replication is completely suppressed with combination antiretroviral therapy (cART). So far, these efforts have not been able to achieve long-term viral suppression or functional cure.

[0015] Two plans to limit or reduce the size of the latent HIV reservoir in people with HIV infection involve (1) intensification (by adding a new ART drug to the patient's treatment regimen) and (2) early treatment (by starting ART immediately after infection). Results from several trials show that the number of HIV-infected cells can be significantly reduced when cART is started in the early acute phase of HIV infection rather than in the chronic late phase.

[0016] In summary, there is a high need for effective and safe drugs for HIV treatment (whether provided as a stand-alone or as an adjunct to cART) that (1) block HIV entry in both cell-free and cell-to-cell modes of transmission, significantly reducing HIV infection of activated or resting CD4 T cells, including those long-lived memory T cells; (2) specifically reactivate HIV-infected resting CD4 T cells to release HIV, leading to apoptosis of latently infected cells; and / or (3) suppress HIV-infected resting CD4 T cell activation / inflammation upon antigen / cytokine stimulation, which occurs at a time that can cause pyroptosis and massive depletion of normal CD4-positive T cells, thus leading to AIDS. An earnest effort for functional or curative cure of HIV infection to result in long-term or permanent remission off cART is an important issue for global public health and is actively being pursued worldwide, and if feasible, this would represent a groundbreaking major change in the treatment of HIV infection.

[0017] References

[0018] Briant, L., Reynes, J., Coudronniere, N., et al. “HIV Reactivation in resting peripheral blood mononuclear cells of infected Adults upon in vitro CD4 cross-linking by ligands of the CDR2-loop in extracellular domain 1.” J. AIDS. 1999. 21 :9-19.

[0019] Briant, L., Coudronniere, N., Robert-Hebmann, V., et al. “Binding of HIV virions or gp120-anti-gp120 immune complexes to HIV-1 infected quiescent peripheral blood mononuclear cells reveals latent infection.” J. Immunol., 1996. 156:3994-4004.

[0020] Burkly, L.C., Olson, D., Shapiro, R., et al. "Inhibition of HIV infection by a novel CD4 domain 2-specific monoclonal antibody. Dissecting the basis for its inhibitory effect on HIV-induced cell fusion." J. Immunol. 1992; 149: 1779-87.

[0021] Carr, F.J., Carter, G., Hamilton, A.A. & Adair, F.S. "Reducing immunogenicity of proteins - by modifying the amino acid sequence of the protein to eliminate potential epitopes for T-cells of a given species." PCT Publication WO 1998-052976.

[0022] Chiba, Y., "Leu3A Binding Peptides." US Patent 5,171,838 (1992).

[0023] Doitsh, G. Galloway, K., Geng, X., et al. "Cell Death by pyroptosis derives CD4 T-cell depletion in HIV infection." Nature. 2014. 505:509-514.

[0024] Global Health Observatory (GHO) HIV / AIDS. http: / / www.who.int / gho / hiv / en /

[0025] HIV-Wikepedia, The free encyclopedia. http: / / en.wikipedia.org / wiki / HIV / AIDS

[0026] Jacobson, J.M. Kuritzkes, D.R., Godofsky, E., et al. “Safety, Pharmacokinetics, and Antiretroviral Activity of Multiple Doses of Ibalizumab (formerly TNX-355), an Anti-CD4 Monoclonal Antibody, in Human Immunodeficiency Virus Type-1-Infected Adults.” Antimicrob. Agents Chemother. 2009. 53:450457.

[0027] Jameson, B.D., Rao, P.E., Kong, L.L. et al. Location and chemical synthesis of a binding site for HIV-1 on the CD4 protein. Science. 1988, 240, 1335-1339.

[0028] Jones, T.D., et al. “Deimmunization of Monoclonal Antibodies.” Methods Mol. Bio. 2009. 525:405-423.

[0029] Kuritzkes, D.R., Jacobson, J.L., Powderly, W.G., et al. “Antiretroviral activity of the anti-CD4 monoclonal antibody TNX-355 in patients infected with HIV type I.” J. Infect. Dis. 2004. 189:286-291.

[0030] Lynn, S. and Wang, C.Y. “Designed deimmunized monoclonal antibodies for protection against HIV exposure and treatment of HIV infection.” US Patent No. 7,501,494 (Issued March 10, 2009).

[0031] Pace, C.S., Fordyce, M.W., Franco, D., et al. “Anti-CD4 Monoclonal Antibody ibalizumab Exhibits Breadth and Potency Against HIV-1, with Natural Resistance Medicated by the loss of a V5 Glycan in Envelope.” J. AIDS. 2013. 62:1-9.

[0032] Pace G, Fordyce M, Franco D. “Anti-CD4 monoclonal antibody ibalizumab exhibits exceptional breadth and potency against HIV, which adopts a unique pathway to resistance.” Abstract 585, 18th CROI 2011, Boston.

[0033] Sawyer, L.S.W., Wrin, M.T., Crawford-Miksza, L., et al. “Neutralization sensitivity of human immunodeficiency virus type 1 is determined in part by the cell in which the virus is propagated.” J. Virol. 1994, 68(3), 1342-1349.

[0034] Sigal, A., Kim, J.T., Balazs, A.B., et al. “Cell-to-Cell spread of HIV permits ongoing replication despite antiretroviral therapy.” Nature. 2011. 477:95-98.

[0035] Than, S., Oyaizu, N., Tetali, S., et al. “Upregulation of human immunodeficiency virus (HIV) replication by CD4 cross-linking on peripheral blood mononuclear cells of HIV-infected adults.” J. Virol. 1997: 71(8):6230-6232.

[0036] Toma, T., Weinheimer, S.P., Stawiski, E., et al.“Loss of Asparagine-linked glycosylation sites in variable region 5 of human immunodeficiency virus type 1 envelope is associated with resistance to CD4 antibodyibalizumab.” J. Virol. 2011. 85:3872-3880

[0037] Wang, C.Y. “Antibodies against a host cell antigen complex for pre and post exposure protection from infection by HIV.” US Patent No. 5,912,176,1999.

[0038] Wang, C.Y., Sawyer, L.S.W., Murthy, K.K., et al. "Postexposure immunoprophylaxis of primary isolates by an antibody to HIV receptor complex." Proc. Nat. Acad. Sci. USA. 1999, 96, 10367-10372. SUMMARY

[0039] The present invention is directed to compositions and methods for the prevention, treatment, and / or functional cure of HIV infection. One aspect of the invention is directed to monoclonal antibodies against CD4, compositions thereof, and methods of using the compositions to prevent, treat, and functionally cure HIV infection.

[0040] One aspect of the invention is directed to antibodies against CD4, compositions thereof, and methods of using the compositions to prevent, treat, and / or functionally cure HIV infection. In certain embodiments, the antibodies can specifically bind to the CDR2 region of domain 1 of CD4. The antibodies of the invention exhibit potent, competitive HIV entry inhibition by their binding to domain 1 of CD4 in both cell-free and cell-cell systems. The antibodies of the invention can also inhibit antigen-induced T cell proliferation and cytokine production (IL2 and IFN-gamma) by CD4-positive T cells, which are involved in the pathogenic cycle of cell necrosis. The antibodies of the invention also have the ability to reactivate silent CD4-positive T cells. This property is particularly useful for the reactivation of latent HIV viral reservoirs in silent T cells, which can make these cells susceptible to antiretroviral drug therapy. To release HIV, such antibodies of high affinity to CD4 can activate silent HIV-infected cells. The reactivation of HIV-infected silent CD4+ T cells allows for the use of combination therapy with the antibodies of the invention and HAART in HIV-infected patients to achieve a functional cure.

[0041] The present invention is directed to methods for the treatment, prevention, and functional cure of HIV infection. In certain embodiments, the dosage form comprises an antibody against CD4. The present invention also comprises antiviral drugs that can be used in methods for the treatment, prevention, and functional cure of HIV infection.

[0042] In certain embodiments, the present application is directed to pharmaceutical compositions comprising monoclonal human, humanized or chimeric, anti-CD4 antibodies having the binding characteristics described above, which when administered as monotherapy at a dose of about 10 mg / kg or higher on a weekly or biweekly schedule, can reduce the viral load of treated subjects to below the limit of detection, and without viral load rebound as long as serum antibody levels are above 10 μg / mL.

[0043] In other embodiments, the present application is directed to pharmaceutical compositions comprising monoclonal humanized anti-CD4 antibodies having the binding characteristics described above, which can serve as a key component of an HAART co-treatment, which when administered to HIV patients not using treatment drugs at a dose of about 10 mg / kg or higher on a weekly or biweekly schedule, will achieve functional cure of the patient. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 is a schematic of competitive and non-competitive HIV entry inhibition mechanisms. FIG. 1A is a graph showing theoretical results provided in a competitive HIV entry inhibition model, where HIV envelope protein gpl20 and inhibitor (e.g. antibody drug) compete for binding to the same site on a common target surface molecule (i.e. CDR2 of CD4 domain 1). In this model, 100% inhibition of HIV binding / entry can be achieved when the inhibitor concentration reaches a certain threshold. In contrast, FIG. 1B is a graph showing theoretical results provided in a non-competitive HIV entry inhibition model, where HIV and inhibitor bind to different sites on the same target molecule (e.g. domain 2 of CD4 for TMB-355). In this non-competitive inhibition model, the inhibitor can reduce HIV binding / entry, but not completely inhibit it regardless of the inhibitor concentration. The "plateau" of inhibition reflects HIV resistance to antibody drugs regardless of drug concentration.

[0045] FIG. 2 is a graph showing results from HIV entry inhibition using TMB-355 on a panel of 118 different HIV-1 Env pseudotyped virus strains covering 11 clades (Pace, G., et al., 2011). For each virus, the black line is the maximum percent inhibition (MPI) when treated with TMB-355 at a concentration of 10 μg / mL (see left Y-axis); and the gray line is the corresponding IC 50 (see right Y-axis). TMB-355 neutralized 92% of the strains with > 50% inhibition, and only 31% of the strains with > 95% inhibition.

[0046] FIG. 3Results from the use of mAb B4 for HIV entry inhibition of a set of over 850 Env-pseudotyped HIV strains collected over a 10-year period. MAb B4 provided broad and potent inhibition of HIV entry with near 100% maximum inhibition percentage (MPI) with all 850 Env-pseudotyped strains and with two IC 50 concentrations (one between 0.01 to 1 pg / mL and the second at about 10 pg / mL).

[0047] FIG. 4 The complete amino acid sequence of the heavy chain of the Fv region of the mAb dB4C7 antibody (SEQ ID NO: 7) used in UB-421. Sequences representing the corresponding CDR1, 2, and 3 regions derived from its parental murine B4 antibody are underlined below (Table 4, SEQ ID NOs: 1 to 3). The variable and constant regions are represented by light and dark gray shading, respectively (SEQ ID NOs: 11 and 12, respectively). The N-glycosylation site at the 101st amino acid residue Asn (N) on the heavy chain is unique to its Fv region location and is important for B4 antibody binding. For the sequence of the Fc region of the substituted human IgGl, the N-glycosylation site at the 298th amino acid residue Asn (N) is removed by substitution with the amino acid His (H) (i.e., N298H). This mutation was found to eliminate IgG-mediated complement-dependent cytotoxicity (CDC) and depletion of CD4-positive T cells in the presence of antibody B4.

[0048] FIG. 5 The complete amino acid sequence of the light chain of the Fv region of the dB4C7 antibody (SEQ ID NO: 8) used in UB-421. Sequences representing the corresponding CDR1, 2, and 3 regions derived from its parental murine B4 antibody are underlined below (Table 4, SEQ ID NOs: 4 to 6). The variable and constant regions are represented by light and dark gray shading, respectively (SEQ ID NOs: 13 and 14, respectively).

[0049] FIG. 6 The complete amino acid sequence of the heavy chain of the further improved humanized antibody (SEQ ID NO: 9) as Met (M) is substituted for Tyr (Y) at the 253rd, 255th, and 257th amino acids of the Fc region of the heavy chain (i.e., M253Y), Ser (S) is substituted for Thr (T) (i.e., S255T), and Thr (T) is substituted for Glu (E) (i.e., T257E), respectively, to provide the antibody with a longer half-life.

[0050] FIG. 7 The complete amino acid sequence of the heavy chain of humanized antibody mAb dB4 (SEQ ID NO: 10) which comprises the variant amino acids discussed in FIG. 4 and 6

[0051] FIG. 8 Figure 3 is a graph showing the binding affinity (antibody concentration) and binding capacity (free and bound antibody) of mAb B4 to surface CD4 located on HPB-ALL cells after three antibody passages on cells.

[0052] FIG. 9 Figure 4 is a graph comparing the binding affinity of mAb B4 (—) and mAb dB4 (—) to soluble CD4 (sCD4) and p2704a peptide capture mix coated on ELISA microtiter plates. Binding affinity was measured using mAb B4 and mAb dB4, respectively, to competitively inhibit B4-biotin and dB4 C7-Alexa binding to the capture mix.

[0053] FIG. 10 Figure 5 is a graph comparing the binding affinity of mAb B4 (—) and mAb dB4 (—) to CD4 located on HPB-ALL cells. Binding affinity was measured by FACS using mAb B4 and mAb dB4, respectively, to competitively inhibit B4-biotin and dB4 C7-Alexa binding to surface CD4 located on HPB-ALL cells.

[0054] FIG. 11 Figure 6 is a graph comparing the binding affinity of mAb dB4 (—) and gpl20MN (—) to CD4 located on HPB-ALL cells. Binding affinity was measured by FACS using mAb dB4 and gpl20MN to competitively inhibit dB4 C7-Alexa binding to surface CD4 located on HPB-ALL cells.

[0055] FIG. 12 Figure 7 is a graph showing the temperature dependent binding (MFI) of mAb dB4 C7 to PBMC CD4+ T cells.

[0056] FIG. 13 ​Graph showing the average percentage (± standard deviation) of unoccupied CD4 receptors (—) and CD4 receptors occupied / bound by mAb dB4 (— ) from blood samples of 6 uninfected individuals as a function of mAb dB4 concentration. Unoccupied receptors were detected using dB4C7-Alexa conjugated to unoccupied binding sites located on the surface of blood CD4+ T cells. However, receptors occupied by mAb dB4 were detected using goat anti-huIgG-FITC.

[0057] FIG. 14 Bar graph showing the effect of the current antiretroviral therapy (cART) drug Tenofovir on HIV cell-free and cell-to-cell transmission (Sigal, A., et al., 2011). The Y axis represents the transmission index from peripheral blood mononuclear cells (PBMCs) isolated from different sources of infection in the presence or absence of tenvofir (Sigal, A. et al., 2011).

[0058] FIG. 15 Bar graph showing viral reactivation in quiescent PBMCs triggered by the following stimuli, measured using HIV-1 p24 gag production, the stimuli include unstimulated (1st bar), phytohemagglutinin (PHA) (2nd bar), inactivated HIV (iHIV) lysate (3rd bar), monoclonal antibody to CDR2 region of CD4 domain 1 (4th bar), monoclonal antibody to CDR3 region of CD4 domain 1 (5th bar), monoclonal antibody to CD4 domain 1 / 2 (6th bar), iHIV in the presence of soluble CD4 (7th bar), monoclonal antibody to CDR2 region of CD4 domain 1 in the presence of soluble CD4 (8th bar), monoclonal antibody to CDR3 region of CD4 domain 1 in the presence of soluble CD4 (9th bar), and monoclonal antibody to CD4 domain 1 / 2 in the presence of soluble CD4 (10th bar), as described in the legend (adapted from Briant L., et al., 1999).

[0059] FIG. 16 Graph showing the competitive inhibition of biotinylated-B4 binding to rsCD4 using anti-HIV RC polyclonal antibodies, measured by ELISA.

[0060] FIG. 17 Graph showing the antibody titration of mAb dB4 and anti-HIV RC polyclonal antibodies on surface CD4 located on PBMCs. Antibody titration is % CD4 binding against antibody concentration (µg / mL).

[0061] FIG. 18A to FIG. 18G This study demonstrates the inhibition of IL-2 and IFN-γ production in proliferating CD4+ and CD8+ T cells induced by the superantigen SEB in treatment-naïve HIV-positive and HIV-negative subjects using mAb dB4 and anti-HIV RC polyclonal antibodies. (For HIV-negative subjects...) FIG. 18A ) and HIV positive ( FIG. 18B The inhibition of IL2 production by proliferating CD4+ T cells induced by superantigen by mAb dB4 and anti-HIV RC polyclonal antibody in HIV-negative subjects is shown in the figure. The figures for HIV-negative subjects and age-matched HIV-positive subjects are also presented. FIG. 18C The inhibition of IL2 production by proliferating CD8+ T cells induced by superantigens by mAb dB4 and anti-HIV RC polyclonal antibodies is also shown in the figure. For HIV-negative ( FIG. 18D ) and HIV positive ( FIG. 18E The inhibition of IFN-γ production by proliferating CD4+ T cells induced by superantigen in HIV-negative subjects is shown in the figure. (Figure 1 shows the effect of mAb dB4 and anti-HIV RC polyclonal antibody on IFN-γ production by superantigen-induced proliferating CD4+ T cells.) FIG. 18F ) and HIV positive ( FIG. 18G The figure shows the inhibition of IFN-γ production by proliferating CD8+ T cells induced by superantigen by mAb dB4 and anti-HIV RC polyclonal antibody in the subjects.

[0062] FIG. 19A to FIG. 19C The results show that by analyzing the primary HIV-1 isolate DH12 (HIV-1) DH12 Pre-exposure ( FIG. 19A ) and post-exposure ( FIG. 19B A diagram illustrating the protection of chimpanzees from HIV infection by administering mAb B4, assessed by measuring HIV-1 RNA copies / mL in PBMCs at a time post-infection. Exposure to HIV-1 DH12 The results of the control group animals that were not given antibodies (Figure) FIG. 19C As shown in the figure. The downward arrow marks the start of the test; at this point, mAb B4 is at HIV-1. DH12 Administer one hour before or after the attack.

[0063] FIG. 20A and FIG. 20B This is a graph showing the HIV viral load in untreated and mAb B4-treated HIV-1-infected chimpanzees, which is assessed by measuring HIV-1 RNA copies / mL over time. FIG. 20ADuration of plasma viremia is compared for chimpanzee X356 (received three infusions of mAb B4 (closed circles)) versus control chimpanzee X084 (received no treatment) who previously received a single dose of mAb B4 in a prior experiment (open circles).

[0064] FIG. 20B Duration of plasma viremia is compared for chimpanzee X356 (received three infusions of mAb B4 (closed circles)) versus control chimpanzee X259 (received no previous dose of mAb B4 (open circles)).

[0065] FIG. 21 Graph comparing binding affinity of mAb dB4 to human (—) and baboon (—) CD4-positive T cells.

[0066] FIG. 22A and FIG. 22B Graph showing the mean Log 10 Graph of change versus days on trial. FIG. 22A Graph showing the viral reduction exhibited by each dose during the trial. FIG. 22B Graph showing the mean and maximum individual nadirs for each dose.

[0067] FIG. 23A to FIG. 23C Graph showing a theoretical comparison of efficacy of UB-421 (mAb dB4C7) and previously reported efficacy data for TMB-355 (TMB-355 was previously known as TNX-355; Kuritzkes, D.R., et al., 2004, Figure 1). FIG. 23A Graph comparing the observed drop in viral load following a single administration of UB-421 at 5 mg / kg and TMB-355 at 3 mg / kg. FIG. 23B Graph comparing the observed drop in viral load following a single administration of UB-421 at 10 mg / kg or TMB-355. FIG. 23C Graph comparing the observed drop in viral load following a single administration of UB-421 at 25 mg / kg or TMB-355.

[0068] FIG. 24A and FIG. 24B Graph showing the mean Log FIG. 24A ) or 25 mg / kg ( FIG. 24BThe mean number of CD4 T cells per PBMC in subjects given UB-421 (mAb dB4C7) was [number] / mm². 3 The diagram shows that the number of stable CD4 T cells in patients treated with UB-421 can be measured using a biotinylated antibody targeting CD4 domain 2.

[0069] FIG. 25A to FIG. 25D This is a graph illustrating the clinical efficacy of UB-421 treatment during the Phase IIa clinical trial, as shown by viral load reduction (top) and the pharmacokinetics of UB-421 (as determined by serum concentration in µg / mL (bottom)). Relevant data are provided for the following representative patients: Patient 1-1-01 who received weekly 10 mg / kg UB-421. FIG. 25A Patient 1-1-02 who received UB-421 at a dose of 10 mg / kg per week FIG. 25B Patient 1-2-03 received UB-421 at 25 mg / kg every two weeks. FIG. 25C ); and patients 1-2-06 who received UB-421 at 25 mg / kg every two weeks. FIG. 25D The entire area shaded in gray represents the duration of UB-421 binding on PBMC CD4+ cells.

[0070] FIG. 26A and FIG. 26B This is a graph showing the theoretical comparison between the viral load reduction observed in the Phase IIa clinical trial using UB-421 and the viral load reduction observed in similar trials of TMB-355 (ibalizumab, formerly known as TNX-355) conducted by others (Jacobson, JM, et al., 2009; Toma, J., et al., 2011; and Pace, CS, et al., 2013). FIG. 26A Overview of viral load changes observed in subjects treated with UB-421 at 10 mg / kg and 25 mg / kg, and FIG. 26B Overview of viral load changes observed in subjects treated with the same dose level of TMB-355.

[0071] FIG. 27 This diagram illustrates the treatment regimen for HIV patients who are not currently using medication or whose HIV is stable on HAART, and who are using UB-421 as a monotherapy alternative to HAART.

[0072] FIG. 28A schematic diagram showing the treatment modalities in a population of HIV patients utilizing UB-421 in combination with HAART therapy to achieve functional cure of HIV infection in HIV patients not using treatment drugs, HIV patients on stable HAART therapy, and HIV patients on failed HAART therapy. DETAILED DESCRIPTION

[0073] The present invention relates to compositions and methods for preventing, treating, and / or functionally curing HIV infection. One aspect of the invention relates to antibodies against CD4, formulations thereof, and methods of using such formulations for preventing, treating, and / or functionally curing HIV infection.

[0074] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references or portions of references cited in this application are expressly incorporated by reference in their entirety for any purpose.

[0075] CD4

[0076] CD4 (cluster of differentiation 4) is a glycoprotein (UniProtKB / Swiss-Prot: P01730.1) that is found on immune cells such as helper T cells, monocytes, macrophages, and dendritic cells (http: / / en.wikipedia.org / wiki / CD4). CD4 is a member of the immunoglobulin superfamily and has four immunoglobulin domains (D1 to D4) exposed on the cell surface. CD4 domains D1 and D3 are similar to immunoglobulin variable (IgV) regions; while D2 and D4 are similar to immunoglobulin constant (IgC) regions. CD4 uses its D1 domain to interact with the β2-domain of class II major histocompatibility complex (MHC II) molecules. Thus, T cells expressing CD4 molecules on their surface are specific for antigens presented by MHC II. A short cytoplasmic / intracellular tail of CD4 containing a special amino acid sequence allows it to interact with the lck molecule.

[0077] The first extracellular domain of CD4 shares homology with immunoglobulins, with three complementarity determining regions (CDRs) similar to those of immunoglobulin chains. Both domain 1 and domain 2 of the extracellular region of the CD4 molecule were found to contribute to the binding site for MHC II molecules; however, domain 1 alone was found to be involved in HIV binding and syncytia formation. In particular, the binding site for the HIV envelope glycoprotein gp120 was found to be located in the CDR2-like loop of domain 1.

[0078] HIV-1 enters the host T cell via its viral envelope protein, called gpl20, by binding to CD4. Binding to CD4 causes a conformational change in gpl20 to allow HIV-1 to bind to the chemokine receptors CCR5 or CXCR4 expressed on the host cell. Then another viral protein, gp41, is structurally altered and HIV inserts a fusion peptide into the host cell to allow fusion of the viral outer membrane with the cell membrane. HIV infection causes a gradual decrease in the number of T cells expressing CD4.

[0079] Antibodies

[0080] One aspect of the present application is directed to antibodies against CD4, compositions thereof, and methods of using the compositions to prevent, treat, and / or functionally cure HIV infection.

[0081] The antibodies of the present application broadly encompass intact antibody molecules, including intact polyclonal, monoclonal, monospecific, multispecific, chimeric, de-immunized, humanized, human, primate, single-chain, single-domain, synthetic and recombinant antibodies, as well as antibody fragments having the desired activity or function.

[0082] The antibodies of the present application can recognize domain 1 of CD4. In some embodiments, the antibodies can specifically bind to the CDR2 region of domain 1 of CD4.

[0083] The antibodies of the present application can be produced by any standard method. In some embodiments, the antibodies of the present application can be produced by immunizing an animal (e.g., mouse, dog, guinea pig, pig, goat, horse, etc.) with a recombinant CD4 protein, a fragment of a recombinant CD4 protein, or a cell expressing CD4 on the surface. Alternatively, the antibodies can be chemically synthesized.

[0084] In some embodiments, the antibodies can be produced by immunizing an animal with a peptide containing the amino acid sequence of domain 1 of CD4. For example, polyclonal antibodies can be produced by immunizing an animal with a peptide containing the amino acid sequence of the CDR2 region of domain 1 of CD4 or a combination thereof. In some embodiments, a peptide containing amino acids 39-66 of CD4 (called the HIV receptor complex ("HIV RC")) is used, as this is the portion of CD4 to which HIV binds. In some embodiments, the HIV RC peptide can be cyclized by disulfide bonds to create a cyclic structure.

[0085] In some embodiments, the cyclic HIV RC peptide can be used to immunize an animal to produce polyclonal antibodies. The "anti-HIV RC polyclonal antibodies" of the present application refer to immune sera against a cyclic peptide containing amino acids 39-66 of the CDR2 region of domain 1 of CD4.

[0086] In other embodiments, the animal is immunized with CD4 positive cells to produce antibodies. For example, in certain embodiments, antibodies are produced by immunizing BALB / c mice with intact, uninfected CD4+human HPB-ALL cells, a T-cell acute lymphoblastic leukemia cell line. This antibody is discussed in more detail in the patents of Wang (U.S. Patents 5,912,176 and 6,090,388) and in the journal article by Wang et al. published in 1999, each of which is incorporated by reference herein in its entirety.

[0087] In other embodiments, the antibodies comprise the amino acid sequences of the heavy and light chains as set forth in the sequence listing. The present application includes homologues and functional analogues of antibodies comprising the amino acid sequences set forth in the sequence listing.

[0088] Functional analogues of the antibodies of the present application include sequence variants and homologues that retain substantially the same functional characteristics (binding recognition, binding affinity, etc.) as the original antibody. For example, antibodies that are variants of the functional analogues or homologues have conservative substitutions at amino acid positions; changes in electrostatic charge; covalent binding to other functional groups; or small additions, insertions, deletions, or conservative substitutions and / or any combination thereof. Thus, the variant antibodies functional analogues and homologues of this antibody will recognize and bind CD4 and be useful in treating HIV in a subject.

[0089] In one embodiment, the antibody functional analogues or homologues generally have at least about 50% sequence similarity to an antibody comprising the amino acid sequences disclosed in the sequence listing. In other embodiments, the antibody functional analogues or homologues have at least about 50%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% similarity to an antibody comprising the amino acid sequences disclosed in the sequence listing.

[0090] A conservative substitution refers to the replacement of one amino acid residue by another with similar chemical properties. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0091] In other embodiments, functional analogs of the antibodies can be modified by addition or deletion of amino acids at the amino or carboxy terminus, and / or by insertion into the sequence. In different embodiments of the application, additions or deletions are made at the amino or carboxy terminus of the peptide. The additions or deletions can be of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues. Such additions or deletions can constitute an amino acid sequence that is not represented in the sequences listed in the sequence listing, but which does not alter the general functional characteristics of the antibody.

[0092] In certain embodiments, the antibodies of the application are labeled or tagged with a chemical substance. For example, the antibodies can be tagged with biotin, a spacer, a probe (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), tetramethylrhodamine isothiocyanate (TRITC), DyLight Fluors, Alexa, green fluorescent protein (GFP), R-phycoerythrin, quantum dots, etc.), an enzyme conjugate, and combinations thereof. In particular embodiments, the antibodies are tagged with biotin or a fluorescent probe.

[0093] In particular embodiments, the antibodies are modified by known deimmunization processes. "Deimmunization" as used herein generally refers to a process used to modify portions of an antibody such that it does not elicit an immune response in an animal when administered to the animal. Specifically, an antibody is immunogenic (e.g., T-cell epitopes) when administered to a particular animal, and deimmunization involves a process of locating and removing portions of the amino acid sequence of the antibody. This process can be achieved by the combined use of immunological and molecular biology techniques. This process has been previously disclosed (e.g., Jones, T.D., et al. 2009). In the context of deimmunization of an antibody, mutations can be introduced generally to remove T-cell epitopes without significantly reducing the binding affinity of the antibody.

[0094] "Humanization" as used herein refers to the modification (deimmunization) of the protein sequence of a non-human species antibody such that immunogenicity is removed when the antibody is administered to a human. In certain embodiments, the antibodies of the application are deimmunized for use in humans using substitution of human constant regions for their constant regions and / or using genes encoding such antibodies expressed in mammalian cells.

[0095] In certain embodiments, the antibodies of the application have heavy and light chain amino acid sequences as shown in Table 4.

[0096] The “mAb B4”, “B4”, or “mouse B4” mentioned in this invention refer to murine monoclonal antibodies having the amino acid sequences of the CDR1, 2, and 3 regions of the heavy and light chains of SEQ ID NOs: 1–6 (as shown in Table 4). These murine monoclonal antibodies recognize CD4 and inhibit HIV entry. The structural and functional characteristics of these antibodies are discussed in more detail in the examples and below.

[0097] The “mAb dB4” or “dB4” mentioned in this invention refers to a human deimmunized antibody derived from mAb B4. Human deimmunized mAb dB4 has the amino acid sequences of the CDR1, 2, and 3 regions of the heavy and light chains of SEQ ID NOs: 1–6 (as shown in Table 4). In some embodiments, the light chain of mAb dB4 has the amino acid sequence of SEQ ID NO: 8 (e.g., ...). FIG. 5 (As shown). In some embodiments, the heavy chain of mAb dB4 has the amino acid sequence of SEQ ID NO: 7 (e.g. FIG. 4 (As shown). In different embodiments, the heavy chain of mAb dB4 has the amino acid sequence of SEQ ID NO: 9 (as shown). FIG. 6 (As shown). MAb B4 can be deimmunized by any suitable method known in the art. In one embodiment, mAb B4 is deimmunized for human use using methods described in U.S. Patents 7,501,494 and 7,872,110, which are incorporated herein by reference in their entirety. In a particular embodiment, human deimmunization of mAb dB4 is achieved by removing the murine antibody constant region (C) of mAb B4. H It is manufactured by replacing the constant region of human IgG1 with Cκ). MAb dB4 comprises dB4 manufactured from any suitable cell clone. In a specific embodiment, mAb dB4 is manufactured by clone 7.

[0098] The “mAb dB4C7” or “dB4C7” mentioned in this invention refers to mAb dB4 expressed by clone 7 containing the recombinant gene B4DIVHv1 / VK1CHO#7, previously disclosed in U.S. Patents 7,501,494 and 7,872,110, which are incorporated herein by reference in their entirety. It shows that clone C7 can produce high-quality mAb dB4 antibodies. Specifically, mAb B4C7 is a human deimmunizing antibody having a light chain containing the amino acid sequence SEQ ID NO: 8 (e.g., ...). FIG. 5 (as shown) and heavy chains containing the amino acid sequence SEQ ID NO: 7 (such as...) FIG. 4The Asn (N) residue at position 298 of mAb dB4C7 was replaced with His (H) to remove the N-glycosylation site, thus eliminating IgG-mediated complement-dependent cytotoxicity (CdC) in the presence of antibody B4 to prevent depletion of CD4-positive T cells.

[0099] "UB-421" as used herein refers to mAb dB4C7 in a suitable form for administration to a human subject.

[0100] The antibodies of the present application can also be described by their interesting and unique functional characteristics.

[0101] For example, the antibodies of the present application can exert potent, competitive HIV entry inhibition by binding to domain 1 of CD4. In particular, the antibodies of the present application have a maximum percent inhibition (MPI) of nearly 100% on all Env-pseudotyped viruses tested, with two IC 50 concentrations; one is between 0.01 and 1 μg / mL, and the second is about 10 μg / mL. The binding activity of the antibodies of the present application is about two logs (i.e., 100-fold tighter binding) higher than the CD4 binding affinity exhibited by the HIV gp120 envelope protein. In addition, the average dissociation constant (Kd) of the antibodies of the present application was measured to be 5.6 x 10 -11 M (range: 3.1 to 8.1 x 10 -11 M), and the maximum binding capacity (Bmax) was measured to be 1.2 x 10 6 Ab (range: 0.93 to 1.4 x 10 6 ).

[0102] The competitive inhibitory properties of the antibodies of the present application were shown in both cell-free and cell-based systems. The antibodies of the present application bind to the CD4 receptor with at least 50-fold higher affinity than the HIV envelope protein gp120 MN. Also, the antibodies of the present application bind to CD4 with higher affinity and specificity than other commercially available antibodies, such as Leu3a.

[0103] The antibodies of the present application also inhibit T cell proliferation and cytokine production (IL2 and IFN-gamma) by CD4 positive T cells in response to antigen, which involves the pathogenic cycle of pyroptosis. Monoclonal antibodies with high affinity for CD4 can inhibit CD4 positive T cell activation and cytokine production (e.g., IL2 and IFN-gamma) in response to antigen (e.g., superantigen SEB (staphylococcal enterotoxin B, SEB)). This antigen-induced activation causes cytokine production by quiescent CD4+ T cells that fail to become infected with HIV, resulting in pyroptosis of these quiescent CD4+ T cells and nearby normal quiescent CD4 positive cells, which leads to massive depletion of CD4+ T cells and thus AIDS.

[0104] The antibodies of the present application also have the ability to reactivate quiescent CD4 positive T cells. This property is particularly useful for reactivating latent HIV viral reservoirs in quiescent T cells, making these cells susceptible to antiretroviral drug therapy. To release HIV, such antibodies with high affinity for CD4 can activate quiescent HIV infected cells. Reactivation of quiescent CD4+ T cells infected with HIV allows the use of a combination therapy of the antibodies of the present application in combination with HAART for the functional cure of HIV infected patients.

[0105] Additional structural and functional features of the antibodies of the present application are provided in the following examples.

[0106] Dosage Form

[0107] The present application is also directed to dosage forms for the prevention, treatment, and / or functional cure of HIV infection. In certain embodiments, the dosage form comprises an antibody against CD4. In particular embodiments, the present application relates to pharmaceutical compositions comprising a monoclonal antibody with high affinity for CD4 (which can be directed to a location within or near the CDR2 region of domain 1 of CD4). The binding activity (EC50) of such antibodies is about two logs (i.e., 100-fold tighter binding) higher than the CD4 binding affinity exhibited by the HIV gp120 envelope protein (EC50 for gp120 = 97 nM). 50 50

[0108] ​​Pharmaceutical formulations of the antibody protein can be prepared by mixing the antibody protein with optional pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include solvents, dispersion media, isotonic agents, and the like. The carrier can be a liquid, a semi-solid (e.g., a paste), or a solid carrier. Examples of carriers include water, saline solution or other buffers (e.g., phosphate, citrate buffer), oils, alcohols, proteins (e.g., serum albumin, gelatin), carbohydrates (e.g., monosaccharides, disaccharides, and other carbohydrates (including glucose, sucrose, trehalose, mannose, mannitol, sorbitol, or dextrins), gels, lipids, liposomes, resins, porous matrices, binders, fillers, coatings, stabilizers, preservatives, antioxidants (including ascorbic acid and methionine), chelating agents (e.g., EDTA); salt forming counter-ions (e.g., sodium); non-ionic surfactants (e.g., TWEEN™, PLURONICS™ or polyethylene glycol (PEG), or combinations thereof.

[0109] The formulation can include at least one active substance. For example, the formulation can contain one or more antibodies for the prevention and treatment of HIV infection and / or one or more compounds with additional advantages. The active substance can be combined with the carrier in any convenient way (e.g., by mixing, dissolving, suspending, encapsulating, entrapping, or the like), and can be formulated into a dosage form suitable for injection, ingestion, infusion, or the like (e.g., tablets, capsules, powders (including lyophilized powders), syrups, suspensions). It can also be formulated as a sustained release formulation.

[0110] In certain embodiments, the pharmaceutical formulation includes mAb dB4C7 for human use. The pharmaceutical formulation including mAb dB4C7 can be formulated in an appropriate buffer, including, but not limited to, citrate, phosphate, Tris, BIS-Tris, and the like, at a pH value between 6.0 and 7.0, and can include excipients (e.g., sugars (50 mM to 500 mM sucrose, trehalose, mannitol, or a mixture thereof), surfactants (e.g., 0.025% - 0.5% Tween 20 or Tween 80), and / or other agents. In specific embodiments, the formulation includes mAb dB4C7 in phosphate buffered saline (PBS) (pH 6.5) containing 20 mM glycine, and 0.05% (v / v) Tween (polysorbate 20). In other specific embodiments, high concentration formulations of mAb dB4 can also be prepared for applications including subcutaneous injection, which include 10 mM histidine.

[0111] Dosage forms containing different amounts of the antibody can be prepared. Generally, a dosage form for administration to a subject contains between about 0.1 mg / mL to about 200 mg / mL. In certain embodiments, a dosage form can contain between about 0.5 mg / mL to about 50 mg / mL; between about 1.0 mg / mL to about 50 mg / mL; between about 1 mg / mL to about 25 mg / mL; or between about 10 mg / mL to about 25 mg / mL of the antibody. In particular embodiments, a dosage form contains about 1.0 mg / mL, about 5.0 mg / mL, about 10.0 mg / mL, or about 25.0 mg / mL of the antibody.

[0112] In particular embodiments, the present application relates to pharmaceutical compositions comprising a human, humanized or chimeric, monoclonal anti-CD4 antibody directed against the CDR2 region of CD4 domain 1 with the binding characteristics described above, which exhibit both competitive HIV entry inhibition and CD4+ T cell activation as an immunotherapy for HIV patients.

[0113] In other embodiments, the present application relates to pharmaceutical compositions comprising a monoclonal human, humanized or chimeric, anti-CD4 antibody with the binding characteristics described above, which as a monotherapy can reduce viral load below the limit of detection in a treated subject whose serum antibody levels are above 10 μg / mL.

[0114] In other embodiments, the present application relates to pharmaceutical compositions comprising a monoclonal human, humanized or chimeric, anti-CD4 antibody with the binding characteristics described above, which as a monotherapy can reduce viral load below the limit of detection in a treated subject whose serum antibody levels are above 10 μg / mL for a 12-week treatment period and maintain stable CD4 T cell numbers.

[0115] In certain embodiments, the present application relates to pharmaceutical compositions comprising a monoclonal human, humanized or chimeric, anti-CD4 antibody with the binding characteristics described above, which as a monotherapy can reduce viral load below the limit of detection in a treated subject for a 12-week treatment period when administered at a dose of about 10 mg / kg or more on a weekly or biweekly schedule.

[0116] In another preferred embodiment, the present application relates to pharmaceutical compositions comprising a monoclonal humanized anti-CD4 antibody with the binding characteristics described above, which as a key component of a co-treatment with HAART can achieve functional cure of HIV patients not using therapeutic drugs when administered at a dose of about 10 mg / kg or more on a weekly or biweekly schedule.

[0117] In another preferred embodiment, the present application relates to a pharmaceutical composition comprising a monoclonal humanized anti-CD4 antibody having the above-mentioned binding characteristics, which can serve as a key component of a co-therapy with HAART, when administered to a patient with stable viral load under HAART at a dose of about 10 mg / kg or higher on a weekly or bi-weekly schedule, will achieve functional cure of the patient.

[0118] Antiviral agent

[0119] The methods of the present application for treating, preventing, and functionally curing HIV infection further comprise an antiviral agent.

[0120] The antiviral agent comprises any agent (chemical or biological) that can effectively inhibit the formation and / or replication of HIV in a mammal. Examples of antiviral agents include, but are not limited to, entry / fusion inhibitors (e.g., maraviroc, enfuvirtide); nucleoside reverse transcriptase inhibitors (NRTIs) and nucleotide reverse transcriptase inhibitors (NtRTIs) (e.g., zidovudine, abacavir, lamivudine, emtricitabine, and tenofovir); non-nucleoside reverse transcriptase inhibitors (NNRTIs) (e.g., nevirapine, efavirenz, etravirine, and rilpivirine); integrase inhibitors (also known as integrase strand transfer inhibitors or INSTIs) (e.g., raltegravir, dolutegravir); protease inhibitors (e.g., saquinavir, saquinavir mesylate, fosamprenavir, tipranavir, lopinavir, indinavir, nelfinavir, amprenavir, ritonavir, darunavir, atazanavir, bevirimat, vivecon); viral maturation inhibitors; agents directed against HIV gene expression; agents directed against important host cell genes and gene products involved in HIV replication; and other anti-HIV agents; iRNA agents; antisense RNA; vectors expressing iRNA agents or antisense RNA; peptide nucleic acids (PNAs); and antiviral antibodies; and combinations thereof.

[0121] Antiviral drugs can be used alone or in combination. The use of antiviral drugs in combination is referred to as antiretroviral therapy (ART), combination antiretroviral therapy (cART), or highly active antiretroviral therapy (HAART). Antiretroviral (ARV) drugs are broadly classified by the stage of the retroviral life cycle that the drug inhibits. Typical combinations include two NRTIs as the "backbone" and one NNRTI, PI, or INSTI as the "base." In certain embodiments, the combination of antiviral drugs is, for example: Combivir, Trizivir, Kaletra, Epzicom, Truvada, Atripla, Complera, Stribild, Triumeq.

[0122] Methods of treatment, prevention, and functional cure

[0123] The present application also includes methods for treating, preventing, and functionally curing HIV infection. In certain embodiments, the dosage form comprises an antibody to CD4.

[0124] In another aspect, the antibody, optionally in a pharmaceutically acceptable carrier, of the present application can be used in a subject to prevent, treat, and / or functionally cure HIV infection, and to prevent transmission of HIV.

[0125] "Treatment" of HIV infection, as described herein, refers to effective inhibition of HIV infection, thereby delaying onset, slowing progression, reducing viral load, and / or ameliorating symptoms caused by HIV infection. Treatment includes pre-exposure and post-exposure.

[0126] "Prevention" of HIV infection, as described herein, refers to delaying the onset of HIV infection, and / or reducing or eliminating the incidence or likelihood of HIV infection. "Prevention" of HIV transmission, as described herein, refers to reducing or eliminating the incidence or likelihood of transmission of HIV from one individual to another (e.g., from an HIV-positive female to a child during pregnancy, childbirth, or breastfeeding).

[0127] "Subject," as described herein, refers to any primate subject, including human, rhesus monkey, baboon, and chimpanzee subjects.

[0128] A therapeutic dose of the antibody of the present application is administered to a subject in need thereof for the treatment and / or prevention of HIV infection.

[0129] A "therapeutically effective amount" as used herein refers to an amount of an antibody effective to inhibit the effects of HIV infection, thereby treating and / or preventing HIV infection. The amount of antibody will depend on the disease state and other clinical factors, such as the weight and condition of the subject, the subject's response to the therapy, the type of dosage form, and the route of administration. The precise amount as a therapeutically effective and non-harmful amount can be determined by one skilled in the art.

[0130] Generally, a suitable dose of antibody for administration to a human adult is in the range of about 3 to 50 mg / kg of the subject's body weight, with a typical initial range of about 5 to 25 mg / kg of the subject's body weight being used. Suitable doses also include about 5.0 mg / kg, about 10.0 mg / kg, or about 25.0 mg / kg of the subject's body weight.

[0131] Therapeutic compositions comprising the human monoclonal antibodies of the present application can be conveniently administered intravenously (e.g., by unit dose injection). Unit dose generally refers to the therapeutic composition of the present application, further referring to a physically discrete unit suitable as unitary dosages to the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic response, in association with the required diluent (i.e., carrier or excipient).

[0132] The compositions are administered in a suitable dosage form and therapeutically effective amount. The amount administered will depend on the subject to be treated, the ability of the active material to reach the site of action, and the degree of therapeutic effect desired. The precise dose to be administered will depend on the judgment of the physician and on the individual's sensitivity to the active ingredient. However, suitable dosage ranges for systemic application disclosed herein depend on the route of administration. Suitable treatment regimens for administration also vary, but are typified by the administration of repeated doses following the initial dose (at intervals of between one or more hours by subsequent injection or other means of administration). Alternatively, continuous intravenous infusion is contemplated to maintain a concentration in the blood within a specified range during the course of the therapy.

[0133] Methods for treating, preventing, and / or functionally curing HIV infection in a subject include administering to the subject an effective amount of a dosage form comprising the antibody. In certain embodiments, the dosage form is provided to the subject in a single administration. In certain embodiments, the dosage form is provided to the subject in multiple administrations. When the dosage form is provided in multiple administrations, the dosage form can be administered once daily, once weekly, once every two weeks (every other week), or once monthly. In particular embodiments, when the treatment schedule is once weekly, the dosage form is administered to the subject at a dose of about 5.0 mg / kg of the subject's body weight. In another embodiment, when the treatment schedule is once every two weeks, the dosage form is administered to the subject at a dose of about 25.0 mg / kg of the subject's body weight.

[0134] In certain embodiments, dosage forms containing the monoclonal antibody exhibit a high safety margin and are well tolerated when repeatedly administered to a subject on a weekly basis at a dose of 5 mg / kg or 25 mg / kg for a total of 8 weeks. In specific embodiments, the monoclonal antibody can be administered to a subject at a dose of 5 mg / kg within hours of an HIV infection to provide a curative treatment of the HIV infection. In other embodiments, the monoclonal antibody can be administered to a subject at a dose of 5 mg / kg within days of an HIV infection to provide a functional treatment of the HIV infection.

[0135] In certain embodiments, the present application relates to pharmaceutical compositions comprising a monoclonal human, humanized or chimeric anti-CD4 antibody having the binding characteristics described above, which can be administered to HIV patients as an immunotherapy to reduce viral load via intravenous or subcutaneous routes of administration. In specific embodiments, the present application relates to pharmaceutical compositions comprising a human, humanized or chimeric monoclonal anti-CD4 antibody directed against the CDR2 region of CD4 domain 1 having the binding characteristics described above, which exhibit competitive HIV entry inhibition and CD4+ T cell activation as an immunotherapy for patients infected with HIV.

[0136] In certain embodiments, the present application relates to pharmaceutical compositions comprising a monoclonal human, humanized or chimeric anti-CD4 antibody having the binding characteristics described above, which can be administered to HIV patients as an immunotherapy to reduce viral load via intravenous or subcutaneous routes of administration on a weekly or biweekly schedule at a dose of about 10 mg / kg or greater.

[0137] In other embodiments, the present application relates to pharmaceutical compositions comprising a monoclonal human, humanized or chimeric anti-CD4 antibody having the binding characteristics described above, which as a monotherapy can increase serum antibody levels in treated subjects to greater than 10 μg / mL, reduce viral load to below the limit of detection.

[0138] In other embodiments, the present application relates to pharmaceutical compositions comprising a monoclonal human, humanized or chimeric anti-CD4 antibody having the binding characteristics described above, which as a monotherapy can increase serum antibody levels in treated subjects to greater than 10 μg / mL, reduce viral load to below the limit of detection.

[0139] In other embodiments, the present application relates to pharmaceutical compositions comprising a monoclonal human, humanized or chimeric anti-CD4 antibody having the binding characteristics described above, which as a monotherapy can increase serum antibody levels in treated subjects to greater than 10 μg / mL, reduce viral load to below the limit of detection.

[0140] In other embodiments, the present application relates to pharmaceutical compositions comprising monoclonal human, humanized or chimeric anti-CD4 antibodies having the binding characteristics described above, which when administered at a dose of about 10 mg / kg or more on a weekly or biweekly schedule as monotherapy, such treatment can reduce the viral load of the treated subject to below the limit of detection and there is no viral load rebound as long as the serum antibody level is above 10 μg / mL.

[0141] In other embodiments, the present application relates to pharmaceutical compositions comprising monoclonal humanized anti-CD4 antibodies having the binding characteristics described above, which can be used as a key component of an adjunct therapy to HAART, which when administered at a dose of about 10 mg / kg or more on a weekly or biweekly schedule to an HIV patient who is not using a therapeutic drug, can achieve functional cure of the patient.

[0142] In other embodiments, the present application relates to pharmaceutical compositions comprising monoclonal humanized anti-CD4 antibodies having the binding characteristics described above, which can be used as a key component of an adjunct therapy to HAART, which when administered at a dose of about 10 mg / kg or more on a weekly or biweekly schedule to a patient who has a stable viral load under HAART, can achieve functional cure of the patient.

[0143] In other embodiments, the present application relates to pharmaceutical compositions comprising monoclonal humanized anti-CD4 antibodies having the binding characteristics described above, which can be administered by intravenous or subcutaneous routes of administration at a dose of about 10 mg / kg or more on a weekly or biweekly schedule to a patient who has failed HAART therapy in an adjunct therapy to HAART, which can further reduce the virus.

[0144] In other embodiments, the present application relates to pharmaceutical compositions comprising monoclonal humanized anti-CD4 antibodies having the binding characteristics described above, which can be administered by intravenous or subcutaneous routes of administration as a key component of a replacement therapy for HAART, whereby a treatment cycle is performed, starting with 2 to 4 months of anti-CD4 antibody treatment, which is followed by a treatment holiday from HAART, as for a patient who has experienced a stable viral load below the limit of detection under HAART, for a period of 1 to 4 or more cycles, which can achieve functional cure.

[0145] In other embodiments, the present application is directed to pharmaceutical compositions comprising monoclonal humanized anti-CD4 antibodies having the binding characteristics described above, which can be administered by intravenous or subcutaneous injection as a key component of HAART replacement therapy, whereby treatment cycles are performed, beginning with 2 to 4 months of anti-CD4 antibody treatment per treatment cycle, as a start for HIV patients not using therapeutic drugs, followed by 2 to 4 months of HAART treatment, for a period of 1 to 4 or more cycles, to achieve functional cure.

[0146] In other embodiments, the present application is directed to pharmaceutical compositions comprising monoclonal humanized anti-CD4 antibodies having the binding characteristics described above, which can be administered by intravenous or subcutaneous injection as a key component of HAART replacement therapy, whereby treatment cycles are performed, beginning with 2 to 4 months of anti-CD4 antibody treatment per treatment cycle, as a start for HIV patients not using therapeutic drugs, followed by 2 to 4 months of HAART treatment, for a period of 1 to 4 or more cycles, to achieve functional cure.

[0147] In other embodiments, the present application is directed to pharmaceutical compositions comprising monoclonal humanized anti-CD4 antibodies having the binding characteristics described above, which can be administered by intravenous or subcutaneous injection as a key component of HAART replacement therapy, whereby treatment cycles are performed, beginning with 2 to 4 months of anti-CD4 antibody treatment per treatment cycle, as a start for HIV patients not using therapeutic drugs, followed by 2 to 4 months of HAART treatment, for a period of 1 to 4 or more cycles, to achieve functional cure.

[0148] In other embodiments, the present application is directed to pharmaceutical compositions comprising monoclonal humanized anti-CD4 antibodies having the binding characteristics described above, which can be administered as a key component of adjunct therapy with HAART, to achieve functional cure of patients not using therapeutic drugs, when administered at a dose of about 10 mg / kg or more on a weekly or biweekly schedule.

[0149] In other embodiments, the present application is directed to pharmaceutical compositions comprising monoclonal humanized anti-CD4 antibodies having the binding characteristics described above, which can be administered as a key component of adjunct therapy with HAART, to achieve functional cure of patients not using therapeutic drugs, when administered at a dose of about 10 mg / kg or more on a weekly or biweekly schedule.

[0150] In other embodiments, the present application relates to pharmaceutical compositions comprising monoclonal humanized anti-CD4 antibodies having the above-described binding characteristics, which can be administered to patients who have failed HAART therapy as adjunct therapy to HAART at a dosage of about 10 mg / kg or more via intravenous or subcutaneous routes of administration on a weekly or biweekly schedule to further reduce viral load.

[0151] In other embodiments, the present application relates to pharmaceutical compositions comprising monoclonal humanized anti-CD4 antibodies having the above-described binding characteristics, which can be administered as adjunct therapy to HAART as a critical component in an intermittent mode for HIV patients not using therapeutic drugs, starting with a treatment period of 2 to 4 months, followed by a treatment holiday of 1 to 2 months, for 1 to 4 or more cycles, to achieve functional cure of the patient as adjunct therapy to the intensive HAART treatment mode.

[0152] In other embodiments, the present application relates to pharmaceutical compositions comprising monoclonal humanized anti-CD4 antibodies having the above-described binding characteristics, which can be administered as adjunct therapy to HAART as a critical component in an intermittent mode for HIV patients not using therapeutic drugs, starting with a treatment period of 2 to 4 months, followed by a treatment holiday of 1 to 2 months, for 1 to 4 or more cycles, to achieve functional cure of the patient as adjunct therapy to the intensive HAART treatment mode.

[0153] In other embodiments, the present application relates to pharmaceutical compositions comprising monoclonal humanized anti-CD4 antibodies having the above-described binding characteristics, which can be administered as adjunct therapy to HAART as a critical component in an intermittent mode for HIV patients not using therapeutic drugs, starting with a treatment period of 2 to 4 months, followed by a treatment holiday of 1 to 2 months, for 1 to 4 or more cycles, to achieve functional cure of the patient as adjunct therapy to the intensive HAART treatment mode.

[0154] In other embodiments, the present application relates to pharmaceutical compositions comprising monoclonal human, humanized or chimeric anti-CD4 antibodies having the above-described binding characteristics, which can be administered to HIV patients as immunotherapy to reduce viral load via intravenous or subcutaneous routes of administration.

[0155] In other embodiments, the present application relates to pharmaceutical compositions comprising a monoclonal human, humanized or chimeric anti-CD4 antibody having the above-described binding characteristics, which can be administered to HIV patients as an immunotherapy to reduce viral load at a dosage of about 5 mg / kg or more via intravenous or subcutaneous injection at a weekly or biweekly schedule. Specific embodiments

[0156] The present application comprises the following specific embodiments:

[0157] (1) A method for treating a subject exposed to HIV infection, comprising: a) administering a pharmacologically effective dose of a monoclonal antibody against CD4 comprising CDRl of the heavy chain of murine antibody B4 of SEQ ID NO: 1, CDR2 of the heavy chain of murine antibody B4 of SEQ ID NO: 2, CDR3 of the heavy chain of murine antibody B4 of SEQ ID NO: 3, CDRl of the light chain of murine antibody B4 of SEQ ID NO: 4, CDR2 of the light chain of murine antibody B4 of SEQ ID NO: 5, and CDR3 of the light chain of murine antibody B4 of SEQ ID NO: 6; b) assessing the level of HIV RNA per milliliter of blood of the subject after step (a).

[0158] (2) The method according to (1), wherein the heavy chain sequence of the antibody comprises SEQ ID NO: 7.

[0159] (3) The method according to (1), wherein the heavy chain sequence of the antibody comprises SEQ ID NO: 9.

[0160] (4) The method according to (1), wherein the light chain sequence of the antibody comprises SEQ ID NO: 8.

[0161] (5) The method according to (1), wherein the heavy chain sequence of the antibody comprises SEQ ID NO: 7, and the light chain sequence of the antibody comprises SEQ ID NO: 8.

[0162] (6) The method according to (1), wherein the heavy chain sequence of the antibody comprises SEQ ID NO: 9, and the light chain sequence of the antibody comprises SEQ ID NO: 8.

[0163] (7) The method according to (1), wherein the administering step (a) is performed within 24 hours of exposure to HIV infection.

[0164] (8) The method according to (1), wherein the administering step (a) is performed within 48 hours of exposure to HIV infection.

[0165] (9) The method according to (1), wherein the pharmacologically effective dose of the monoclonal antibody is administered at serum levels of about 10 μg / ml or greater for a period of 12 weeks on a weekly or biweekly schedule.

[0166] (10) The method according to (1), wherein a value of HIV RNA levels per milliliter of less than 1 copy / ml is considered viral eradication.

[0167] (11) The method according to (1), wherein a value of HIV RNA levels per milliliter of between 1 and less than 50 copies / ml is considered functional cure of the virus.

[0168] (12) A method for treating an HIV patient comprising: a) administering a pharmacologically effective dose of a composition comprising a monoclonal antibody against CD4 comprising CDR1 of the heavy chain of murine antibody B4 of SEQ ID NO: 1, CDR2 of the heavy chain of murine antibody B4 of SEQ ID NO: 2, CDR3 of the heavy chain of murine antibody B4 of SEQ ID NO: 3, CDR1 of the light chain of murine antibody B4 of SEQ ID NO: 4, CDR2 of the light chain of murine antibody B4 of SEQ ID NO: 5, and CDR3 of the light chain of murine antibody B4 of SEQ ID NO: 6; and highly active antiretroviral therapy (HAART); and b) assessing the HIV RNA levels per milliliter in the blood of the subject after step (a).

[0169] (13) The method according to (12), wherein the heavy chain sequence of the antibody comprises SEQ ID NO: 7.

[0170] (14) The method according to (12), wherein the heavy chain sequence of the antibody comprises SEQ ID NO: 9.

[0171] (15) The method according to (12), wherein the light chain sequence of the antibody comprises SEQ ID NO: 8.

[0172] (16) The method according to (12), wherein the heavy chain sequence of the antibody comprises SEQ ID NO: 7 and the light chain sequence of the antibody comprises SEQ ID NO: 8.

[0173] (17) The method according to (12), wherein the heavy chain sequence of the antibody comprises SEQ ID NO: 9 and the light chain sequence of the antibody comprises SEQ ID NO: 8.

[0174] (18) The method according to (12), wherein the pharmacologically effective dose of the antibody is administered at a dose of 10 mg / kg or more on a weekly or biweekly basis.

[0175] (19) The method according to (12), wherein the antibody is administered in an intermittent mode as an adjunct therapy in a HAART treatment regimen.

[0176] (20) The method according to (19), wherein each cycle is administration of the antibody as an adjunct therapy in a HAART treatment regimen for a period of about 2 to 4 months, followed by a 1 to 2 month holiday from the antibody in the HAART treatment regimen.

[0177] (21) The method according to (20), wherein the intermittent mode is continued for a period of one to four cycles.

[0178] (22) A method for treating an HIV patient, comprising: a) reducing the latent HIV viral reservoir in the HIV patient by activating HIV viral expression and apoptosis of latently infected cells in the patient; and b) administering to the patient a pharmacologically effective dose of HAART.

[0179] (23) The method according to (22), wherein activating HIV viral expression and apoptosis of latently infected cells in the patient is performed by administering to the patient a pharmacologically effective dose of a monoclonal antibody to CD4 comprising CDR1 of the heavy chain of murine antibody B4 of SEQ ID NO: 1, CDR2 of the heavy chain of murine antibody B4 of SEQ ID NO: 2, CDR3 of the heavy chain of murine antibody B4 of SEQ ID NO: 3, CDR1 of the light chain of murine antibody B4 of SEQ ID NO: 4, CDR2 of the light chain of murine antibody B4 of SEQ ID NO: 5, and CDR3 of the light chain of murine antibody B4 of SEQ ID NO: 6.

[0180] (24) The method according to (22), wherein activating HIV viral expression and apoptosis of latently infected cells in the patient is performed by administering to the patient a histone deacetylase (HDAC) inhibitor.

[0181] (25) A method for treating a subject exposed to HIV infection, comprising: a) administering a pharmacologically effective dose of a monoclonal antibody having a high affinity for the CD4 CDR2-like domain region; and b) assessing the level of HIV RNA per milliliter of blood of the subject after step (a).

[0182] (26) The method according to (25), wherein the heavy chain sequence of the antibody comprises SEQ ID NO: 7.

[0183] (27) The method according to (25), wherein the heavy chain sequence of the antibody comprises SEQ ID NO: 9.

[0184] (28) The method according to (25), wherein the light chain sequence of the antibody comprises SEQ ID NO: 8.

[0185] (29) The method according to (25), wherein the heavy chain sequence of the antibody comprises SEQ ID NO: 7, and the light chain sequence of the antibody comprises SEQ ID NO: 8.

[0186] (30) The method according to (25), wherein the heavy chain sequence of the antibody comprises SEQ ID NO: 9, and the light chain sequence of the antibody comprises SEQ ID NO: 8.

[0187] (31) The method according to (25), wherein the administering step (a) is performed within 24 hours of exposure to HIV infection.

[0188] (32) The method according to (25), wherein the administering step (a) is performed within 48 hours of exposure to HIV infection.

[0189] (33) The method according to (25), wherein the pharmacologically effective dose of the monoclonal antibody is administered at a serum level of about 10 μg / ml or greater on a weekly or biweekly schedule for a period of 12 weeks.

[0190] (34) The method according to (25), wherein a value of HIV RNA levels per milliliter of less than 1 copy / ml is considered eradication of the virus.

[0191] (35) The method according to (25), wherein a value of HIV RNA levels per milliliter of between 1 and less than 50 copies / ml is considered functional cure of the virus.

[0192] Additional embodiments

[0193] (1) A method for treating a subject exposed to HIV, comprising administering to the subject a pharmacologically effective dose of an antibody directed to CD4 domain 1.

[0194] (2) The method according to (1), wherein the antibody specifically binds to a CDR2 region located in CD4 domain 1.

[0195] (3) The method according to (2), wherein the antibody is a monoclonal antibody, a polyclonal antibody, or a combination thereof.

[0196] (4) The method according to (2), wherein the antibody is a humanized monoclonal antibody.

[0197] (5) The method according to (4), wherein the humanized monoclonal antibody comprises: a heavy chain amino acid sequence comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; and a light chain amino acid sequence comprising CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6.

[0198] (6) The method according to (4), wherein the humanized monoclonal antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 11; and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0199] (7) The method according to (4), wherein the humanized monoclonal antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 10; and a light chain comprising the amino acid sequence of SEQ ID NO: 8.

[0200] (8) The method according to (7), wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 7.

[0201] (9) The method according to (8), wherein the humanized antibody is administered to the subject prior to exposure to HIV.

[0202] (10) The method according to (8), wherein the humanized antibody is administered to the subject after exposure to HIV.

[0203] (11) The method according to (10), wherein the humanized antibody is administered within 48 hours after exposure to HIV.

[0204] (12) The method according to (8), wherein the humanized antibody is administered to the subject at a dose of at least about 5 mg / kg body weight.

[0205] (13) The method according to (12), wherein the humanized antibody is administered to the subject in multiple doses.

[0206] (14) The method according to (13), wherein the humanized antibody is administered to the subject at between once a week or once every two weeks.

[0207] (15) The method according to (13), further comprising the step of administering to the subject an antiviral drug.

[0208] (16) The method according to (15), wherein the antiviral drug is highly active antiretroviral therapy (HAART).

[0209] (17) The method according to (16), wherein the HAART comprises a nucleoside analog reverse transcriptase inhibitor in combination with a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor.

[0210] (18) The method according to (16), wherein the humanized antibody is administered concurrently with the HAART.

[0211] (19) The method according to (16), wherein the humanized antibody and the HAART are administered to the subject for a cycle, wherein the cycle comprises: (i) administering the humanized antibody to the subject at between every week or every two weeks for a period of 4 months, followed by a two-month treatment holiday; and (ii) continuing to administer the HAART to the subject for the six-month period of (i).

[0212] (20) The method according to (18), wherein the subject is treated for two cycles.

[0213] (21) A method for treating a subject infected with HIV, comprising administering to the subject a treatment regimen comprising: (a) a pharmacologically effective dose of an antibody directed against CD4 domain 1 ; and (b) a highly active antiretroviral therapy (HAART).

[0214] (22) The method according to (21), wherein the antibody specifically binds to a CDR2 region located in CD4 domain 1.

[0215] (23) The method according to (22), wherein the antibody is a monoclonal antibody, a polyclonal antibody, or a combination thereof.

[0216] (24) The method according to (22), wherein the antibody is a humanized monoclonal antibody.

[0217] (25) The method according to (24), wherein the humanized monoclonal antibody comprises: a heavy chain amino acid sequence comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 3; and a light chain amino acid sequence comprising a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 5, and a CDR3 of SEQ ID NO: 6.

[0218] (26) The method according to (24), wherein the humanized monoclonal antibody comprises: a heavy chain comprising an amino acid sequence of SEQ ID NO: 11; and a light chain comprising an amino acid sequence of SEQ ID NO: 13.

[0219] (27) The method according to (24), wherein the humanized monoclonal antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 10; and a light chain comprising the amino acid sequence of SEQ ID NO: 8.

[0220] (28) The method according to (27), wherein the humanized antibody is administered to the subject at a dose of at least about 5 mg / kg body weight.

[0221] (29) The method according to (21), wherein the treatment regimen is administered to the subject over a cycle, wherein the cycle comprises: (i) administering the humanized antibody to the subject over a period of between every week or every two weeks for a period of four months, followed by a two month treatment holiday; and (ii) administering HAART to the subject continuously over the six month period of (i).

[0222] (30) The method according to (18), wherein the subject is treated with two cycles.

[0223] Further embodiments

[0224] (1) A composition for treating a subject exposed to HIV comprising: a pharmacologically effective amount of an antibody directed to CD4 domain 1.

[0225] (2) The composition according to (1), wherein the antibody specifically binds to a CDR2 region located in CD4 domain 1.

[0226] (3) The composition according to (2), wherein the antibody is a monoclonal antibody, a polyclonal antibody, or a combination thereof.

[0227] (4) The composition according to (2), wherein the antibody is a humanized monoclonal antibody.

[0228] (5) The composition according to (4), wherein the humanized monoclonal antibody comprises: a heavy chain amino acid sequence comprising: a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 3; and a light chain amino acid sequence comprising: a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 5, and a CDR3 of SEQ ID NO: 6.

[0229] (6) The composition according to (4), wherein the humanized monoclonal antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 11; and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0230] (7) The composition according to (4), wherein the humanized monoclonal antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 10; and a light chain comprising the amino acid sequence of SEQ ID NO: 8.

[0231] (8) The composition according to (7), wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 7.

[0232] (9) The composition according to (8), wherein the humanized antibody is administered to the subject prior to exposure to HIV.

[0233] (10) The composition according to (8), wherein the humanized antibody is administered to the subject after exposure to HIV.

[0234] (11) The composition according to (10), wherein the humanized antibody is administered within 48 hours after exposure to HIV.

[0235] (12) The composition according to (8), wherein the humanized antibody is administered to the subject at a dose of at least about 5 mg / kg body weight.

[0236] (13) The composition according to (12), wherein the humanized antibody is administered to the subject multiple times.

[0237] (14) The composition according to (13), wherein the humanized antibody is administered to the subject at intervals of between once a week or once every two weeks.

[0238] (15) The composition according to (13), wherein the subject is treated (or administered) with an antiviral drug.

[0239] (16) The composition according to (15), wherein the antiviral drug is highly active antiretroviral therapy (HAART).

[0240] (17) The composition according to (16), wherein the HAART comprises a nucleoside analogue reverse transcriptase inhibitor in combination with a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor.

[0241] (18) The composition according to (16), wherein the humanized antibody is administered concurrently with the HAART.

[0242] (19) The composition according to (16), wherein the humanized antibody and the HAART are administered to the subject for a cycle, wherein the cycle comprises: (i) administering the humanized antibody to the subject at intervals of between once a week or once every two weeks for a period of 4 months, followed by a two-month treatment holiday; and (ii) continuously administering the HAART to the subject for a period of six months within (i).

[0243] (20) The composition according to (18), wherein the subject is treated for a period of two cycles.

[0244] (21) A composition for treating a subject infected with HIV comprising: (a) a pharmacologically effective dose of an antibody directed against CD4 domain 1; and (b) highly active antiretroviral therapy (HAART).

[0245] (22) The composition according to (21), wherein the antibody specifically binds to a CDR2 region located in CD4 domain 1.

[0246] (23) The composition according to (22), wherein the antibody is a monoclonal antibody, a polyclonal antibody, or a combination thereof.

[0247] (24) The composition according to (22), wherein the antibody is a humanized monoclonal antibody.

[0248] (25) The composition according to (24), wherein the humanized monoclonal antibody comprises: a heavy chain amino acid sequence comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 3; and a light chain amino acid sequence comprising a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 5, and a CDR3 of SEQ ID NO: 6.

[0249] (26) The composition according to (24), wherein the humanized monoclonal antibody comprises: a heavy chain comprising an amino acid sequence of SEQ ID NO: 11; and a light chain comprising an amino acid sequence of SEQ ID NO: 13.

[0250] (27) The composition according to (24), wherein the humanized monoclonal antibody comprises: a heavy chain comprising an amino acid sequence of SEQ ID NO: 10; and a light chain comprising an amino acid sequence of SEQ ID NO: 8.

[0251] (28) The composition according to (27), wherein the humanized antibody is administered to the subject at a dose of at least about 5 mg / kg body weight.

[0252] (29) The composition according to (21), wherein a treatment regimen is administered to the subject for a period of a cycle, wherein the cycle comprises: (i) administering the humanized antibody to the subject for a period of four months at either weekly or biweekly intervals, followed by a two month treatment holiday; and (ii) continuously administering the HAART to the subject for a period of six months during (i).

[0253] (30) The composition according to (18), wherein the subject is treated for two cycles.

[0254] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. As used herein, the articles "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Similarly, the word "or" is intended to mean "and" unless the context clearly indicates otherwise. Thus, for example, the phrase "comprising A or B" is intended to encompass the embodiments comprising A, B, or both A and B. It is further to be understood that all amino acid sequence sizes and all molecular weight or mass values are approximate, and are given as descriptive. However, methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods, or in the practice of the methods and materials presented throughout this disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including the

[0255] The following explanatory discussion of the drawings and associated embodiments is provided to facilitate an understanding of certain terminology frequently used herein, particularly in the embodiments. The discussion is provided for convenience and is not intended to limit the application.

[0256]

EMBODIMENT

[0257] Example 1. Immunization and functional properties of MAb B4

[0258] Monoclonal antibody B4 (mAb B4) is a monoclonal antibody that recognizes a complex HIV receptor site (CD4) on the surface of T cells. MAb B4 can affect and interfere with the interaction of CD4 and HIV co-receptors. MAb B4 preferentially neutralizes primary HIV-1 isolates.

[0259] The following information summarizes the discovery and initial characterization of murine mAb B4, including information from two patents (U.S. Patents 5,912,176 and 6,090,388, both invented by Wang) and a journal article by Wang et al. (1999), which are incorporated by reference in their entirety.

[0260] 1. Murine monoclonal antibody derived from immunization of HPB-ALL

[0261] BALB / c mice were immunized with intact, uninfected CD4+ human HPB-ALL cells, a T cell acute lymphoblastic leukemia cell line, to provide mAb B4.

[0262] A new class of anti-CD4 antibodies (designated mAb B4) is provided which is specific for CD4 located on the cell surface and has broad neutralizing activity against primary isolates of HIV-1.

[0263] 2. Epitope recognized by MAb B4

[0264] MAb B4 was found to preferentially recognize membrane-bound CD4 located on the cell surface as compared to recombinant soluble CD4 (rsCD4).

[0265] MAb B4 binding to membrane-bound CD4 prior to exposure to HIV was shown to block subsequent gpl20 and whole virus attachment to CD4. However, membrane-bound CD4 bound to gpl20 prior to exposure to the antibody was still able to bind MAb B4. Thus, MAb B4 was able to affect gpl20 binding to membrane-bound CD4, but gpl20 did not affect MAb B4 binding to CD4.

[0266] The epitope recognized by MAb B4 is distinct from the epitopes recognized by other well-studied anti-CD4 monoclonal antibodies, including the monoclonal antibodies Leu3a and OKT4A (Chiba, Y. 1992; Jameson, B.D., et al., 1988) which recognize CD4 domain 1, and the monoclonal antibody 5A8 (Burkly, et al., 1992) which recognizes CD4 domain 2.

[0267] 3. In vitro neutralizing activity of MAb B4

[0268] By general definition, murine MAb B4 is not a neutralizing antibody. Rather, MAb B4 inhibits viral entry by covering the host cell receptor, rather than by attaching to the virus. The effect of MAb B4 on HIV infection can be readily observed by viral neutralization assays used in the field (e.g., the MT-2 Microplaque Neutralization Assay (Sawyer et al., 1994)). The neutralizing activity of murine MAb B4 was assessed by our collaborator, Dr. Carl Hanson (California Department of Health Services), and independently in the laboratories of Dr. John Mascola (Henry Jackson Foundation, WRAIR), Dr. David Montefiori (Duke University), and Dr. Malcolm Martin (NIAID). The following HIV neutralization properties were extensively characterized in relation to MAb B4 between 1995 and 2010:

[0269] (1) PBMC-grown primary isolates (PBMC-grown primary isolates) are more susceptible to HIV-1 than T cell line adapted isolates IIIB and HIV-1 MN more sensitive to neutralization by mAb B4.

[0270] (2) MAb B4 neutralizes infection by primary isolates that use both CCR5 / CXCR4 (dualtropic) and CCR5 coreceptors.

[0271] (3) MAb B4 has low activity against T cell line adapted HIV-1 isolates that use CXCR4 coreceptors.

[0272] (4) MAb B4 neutralizes primary isolates representing different syncytial inducing (SI) and non-syncytial inducing (NSI) HIV-1 subtypes A-G to 90% endpoints and up to 3 logs of infectivity.

[0273] (5) MAb B4 neutralizes HIV-2, simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) with dualtropic coreceptors in the HIV-1 envelope.

[0274] (6) In tonsil tissue systems, mAb B4 reduces infectivity of HIV-1 primary isolate VL135 (HIV-1 VL135 ) by two logs. In the presence of activated human complement, where many anti-viral antibodies exhibit antibody-dependent enhancement, as little as 12.5 μg / mL of mAb B4 completely neutralized >100 TID 50 (50% tonsil infectious dose) of the monocytotropic isolate JR-CSF.

[0275] (7) mAb B4 exhibits neutralizing activity against HIV-1 VL135 when added up to 48 hours post-infection; significant anti-viral effects are seen when mAb B4 is added up to 72 hours post-infection.

[0276] a. It is equally effective whether or not pre-incubated with cells or virus.

[0277] b. Its effect is by blocking the spread of the infection to new cells, rather than by post-entry mechanisms.

[0278] c. In these assays, mAb B4 does not contribute to cytotoxicity.

[0279] Example 2. HIV-1 neutralization and drug resistance testing

[0280] Viral neutralization and drug resistance testing on multiple HIV isolates from different clades was performed in the laboratories of Dr. Carl Hanson and Monogram Biosciences from 1998 to 2011. Detailed descriptions of the tests are described below.

[0281] 1. HIV-1 neutralization testing

[0282] Blood or antibody samples were collected as indicated for each test. Serum or antibody samples were evaluated on a panel of HIV-1 isolates containing multiple clades using the MT-2 microplaque test or the mitogen (PHA)-stimulated PBMC test.

[0283] 1.1. MT-2 microplaque test

[0284] The MT-2 microplaque test is limited to syncytium-inducing isolates of HIV. The test is performed in 96-well plates in which up to 25 microplaques per well can be counted by fluorescent staining of syncytia on the microplaques. In this test, infected MT-2 cells are layered by centrifugation through molten agarose, which solidifies during centrifugation. The test is considered sensitive and has a dynamic range spanning several orders of magnitude. The test is also considered the only efficient method for processing large numbers of samples. Computerized statistical analysis of large numbers of replicates is used to provide a degree of quality control and standardization that is difficult to achieve using other methods.

[0285] 1.2. PBMC test

[0286] The PBMC test is a standard antigen-reduction test in which p24 antigen expression is quantitated in PBMCs by antigen-capture ELISA after infected cells have been grown in 96-well microplates. The advantage of this test is that it is applicable to all HIV strains and isolates.

[0287] 1.3. Virus stocks

[0288] HIV stocks used for in vitro and in vivo neutralization tests are listed in Tables 3, 5 and 6, and FIG. 1A , 1B, 3, 22, and 23. Primary HIV-1 viruses from subtypes A through G and H were: (a) isolated from men who have sex with men participating in the San Francisco Men's Health Study at the Virus and Rickettsial Disease Laboratory (VRDL) of the California Department of Health Services; (b) obtained from the World Health Organization network of HIV isolation and characterization, (c) provided by the U.S. Military HIV Research Program, and (d) donated by the National Institute of Allergy and Infectious Diseases AIDS Research and Reference Reagent Program. DH-12 (isolated from a patient, passaged in chimpanzee peripheral blood mononuclear cells (PBMC)) was also provided by the National Institute of Allergy and Infectious Diseases AIDS Research and Reference Reagent Program.

[0289] 1.4. B4 or dB4 neutralization activity

[0290] B4 or dB4 neutralization activity is defined as the concentration of antibody that provides a specified percentage (50-95%) of virus reduction relative to a control group containing no antibody. The concentration of antibody at the 50% and 90% endpoint indicators is derived by interpolation between antibody dilutions.

[0291] 2. PhenoSense HIV entry assay

[0292] The PhenoSense HIV entry assay to determine drug resistance was performed at Monogram Biosciences (South San Francisco, CA).

[0293] Cells are infected with recombinant virus produced from the vector library in the presence of different concentrations of drug or antibody (e.g., B4 or dB4). The amount of drug required to inhibit viral replication of the test vector is determined at 50% (IC 50 ) or 90% (IC 90 ).

[0294] 2.1. Generation of recombinant viruses used in the PhenoSense HIV assay

[0295] The recombinant viruses used in the PhenoSense HIV assay are derived from patient samples that are selected in a longitudinal study of HIV infection and are determined to be HIV seropositive. For individuals who become HIV infected, clinical and plasma samples are collected for laboratory evaluation, including HIV viral load and CD4 cell counts. For individuals who are initially seronegative but become seropositive within the following year, HIV infection is confirmed using two enzyme immunoassays and western blot.

[0296] Samples from subjects with subtypes A, BF, C, D, E, EA, F, G, or J during seroconversion were collected (based on previous HIV subtype analysis, which was performed using hybridization assays) to construct recombinant viruses (as shown in Table 3). The HIV env, pol regions were amplified from the test samples, and the amplified DNAs were cloned into test vectors. The vector pools were sequenced at GeneSeq HIV to determine the HIV genotype. In the PhenoSense HIV test, cells were infected with recombinant viruses generated from the vector pools in the presence of different drug concentrations.

[0297] Example 3. Neutralizing activity of MAb B4 against HIV isolates of all clades (using the PhenoSense test from Monogram Biosciences)

[0298] It was demonstrated that mAb B4 can neutralize all HIV viruses of clade B. In this test, a total of 73 representative non-B clade HIV isolates were made into recombinant viruses from clade A (n=8), BF (n=l), C (n=18), D (n=18), E (n=4), EA (n=10), F (n=8), G (n=4), J (n=2), plus three control viruses 92HT594, JRCSF, JRFL, and tested in the PhenoSense HIV test to analyze their sensitivity to mAb B4 (see Table 3). It was shown that all recombinant viruses were highly sensitive to mAb B4 with unprecedented low IC 50 and IC 90 concentrations with an average IC 50 = 0.018 µg / mL and IC 90 = 0.062 µg / mL. It is noteworthy that many of these HIV virus isolates were found to be derived from patients with multiple drug resistance, which clearly indicates that mAb B4 or its human counterpart would be highly effective in the treatment of patients who have developed resistance to HIV drugs.

[0299] Example 4. Competitive HIV entry inhibition by monoclonal antibody B4: prevention of the unexpected effect of post-treatment HIV resistant mutants

[0300] A competitive inhibition test can assess the ability and efficacy of an inhibitor (e.g., an entry inhibitor antibody) to compete with HIV envelope protein for the same receptor binding site on CD4 to inhibit HIV entry into cells. In a theoretical test, mAb B4 and HIV envelope protein (gpl20) compete for binding to CD4. FIG. 1AThe predicted results of this experiment are shown, where each line represents a different viral isolate. Specifically, the expected results from this theoretical experiment demonstrate that while different viral isolates have different sensitivities to mAb B4 (IC 50 ), as long as mAb B4 is present at a sufficient concentration to inhibit entry of all viral isolates by 100%, the maximum inhibition percentage (MPI) is 100%.

[0301] By comparison, non-competitive inhibition assays can assess the ability and efficacy of an inhibitor (e.g., a co-receptor antagonist or an antibody that binds to a different portion of CD4) to inhibit or reduce the binding of HIV envelope proteins to CD4, thereby inhibiting HIV entry into a cell. In a theoretical experiment, the ability of a non-competitive inhibitor (e.g., TMB-355) to inhibit the binding of HIV envelope proteins (gpl20) to CD4 was analyzed. FIG. 1B The predicted results of this experiment are shown, where each line represents a different viral isolate. Specifically, the expected results from this theoretical experiment demonstrate that different viral isolates have different sensitivities to TMB-355 (IC 50 ), and at least some fraction of viral isolates can enter cells regardless of the amount of TMB-355 present. Based on this theoretical experiment, a “plateau” of maximum inhibition percentage (MPI) can be expected, where regardless of IC 50 HIV resistance can be observed.

[0302] TMB-355 (formerly TNX-355, also known as Ibalizumab) is a humanized IgG4 monoclonal antibody designed to bind to the extracellular domain 2 of both rhesus and human CD4 to prevent HIV from binding and entering CD4+ cells after binding (e.g., Burkly, LC, et al., 1992; and Kurizkes, DR, et al., 2004). The TMB-355 antibody binding site on CD4 is distinct from the site required for HIV envelope protein gpl20 binding and from the site required for interaction with major histocompatibility complex proteins. Accordingly, TMB-355 mediates non-competitive HIV entry inhibition.

[0303] TMB-355 has been shown to have potent neutralizing activity against some HIV-1 viruses, but its inhibitory activity was not consistent with its neutralizing activity when evaluated against a broad panel of HIV-1 strains. FIG. 2 The maximum inhibition percentage (MPI) of TMB-355 was shown to range from 100% to 15% (left Y-axis) with increasing IC 50Figure 6. HIV entry inhibition by mAb B4. The Y axis (right) shows the percent inhibition of HIV entry by mAb B4. Each bar represents an HIV isolate from a group of 118 Env pseudotyped HIV viruses (Song, R., et al., 2013). Of all clades analyzed, clades A and E viruses were significantly more sensitive to TMB-355 than non-clade A and E viruses. In addition, it was found that viral resistance mutations in the V5 region of gpl20 were found in patients receiving TMB-355 treatment to reduce viral load (Toma, J., et al., 2011; Pace, C.S., et al., 2013). The non-competitive inhibitory effect demonstrated by TMB-355 (Ibalizumab) shows a high likelihood of developing resistant HIV mutants during antibody treatment, as isolates with less than 100% inhibition will undergo viral replication.

[0304] In contrast, results from a group of over 850 Env pseudotyped HIV viruses collected over a 10 year period show that mAb B4 provides unexpected breadth and potency of HIV entry inhibition FIG. 3 From these collected data, it can be seen that mAb B4 has a near 100% maximum percent inhibition (MPI) with two IC 50 concentrations, one between 0.01 and 1 μg / mL and the other at about 10 μg / mL. The HIV entry inhibition curve for mAb B4 has the typical characteristics of a competitive inhibitory mechanism, as it has an MPI of ~100% for each HIV virus. Given the significantly strong competitive HIV entry inhibition characteristics of mAb B4, it is unlikely that viral resistance mutants will develop during mAb B4 treatment. This tight competitive inhibition, as exhibited by mAb B4, has never been observed with any other tested HIV inhibitor to date. 50

[0305] The MPI and IC 50 values from this example, combined with data showing that multiple HIV isolates derived from multiply resistant patients are highly sensitive to mAb B4 (Example 3), show that mAb B4 or its human counterpart will be highly effective in the treatment of resistant HIV patients who have failed HAART therapy. The neutralization pattern mediated by mAb B4 provides a unique HIV drug that can prevent the generation of resistant viral mutants in HIV patients receiving mAb B4 or its human counterpart analog (with similar Fv region) treatment.

[0306] Example 5. Humanization of Monoclonal Antibody B4

[0307] ​Because the murine antibody, mAb B4, is immunogenic in humans, humanization of the murine antibody can be achieved through a process of known deimmunization techniques (Jones, T.D., et al. 2009). Deimmunization of mAb B4 is described in detail in U.S. Patent No. 7,501,494 to Lynn., S. and Wang, C.Y. (both of which are incorporated by reference herein in their entireties), as outlined below.

[0308] First, the constant regions (C H and Ck) of the murine antibody B4 were completely removed and replaced with the constant regions of human IgGl (SEQ ID NOs: 12 and 14, respectively), while retaining the Fv portion, to create a chimeric B4 antibody. Next, the Fv fragment of the murine mAb B4 was deimmunized for human use by identifying and removing murine T- and B-cell epitopes that are potentially immunogenic. The T-cell epitopes were removed after identifying such epitopes in the variable regions of mAb B4. The amino acid sequences of the variable regions were analyzed using a three-dimensional "peptide threading" method to present MHC class II-binding motifs. Removal of the B-cell epitopes from the variable regions was achieved by "cloaking" of surface residues without interfering with antibody recognition. This deimmunized, humanized form of mAb B4 is referred to as mAb dB4.

[0309] U.S. Patent No. 7,501,494 to Lynn., S. and Wang, C.Y. discusses that IgGl contains a biantennary complex N-terminal binding carbohydrate in the CH2 region, which is important for effector functions, such as complement fixation and antigen-dependent-cellular-cytotoxicity (ADCC), which can result in elimination of the target antigen. Because mAb B4 acts on the CD4 receptor complex, it can cause destruction of CD4+ cells and immunosuppression of CD4+ cell function through the effector functions of IgGl responsible for binding complement. Therefore, removal of the N-glycosylation site located in the Fc region of IgGl can disrupt the ability of IgGl to bind to human FcRl to activate complement, or bind Clq, thereby eliminating IgG-mediated complement-dependent cytotoxicity (CdC). Removal of the N-glycosylation site located in the Fc region of IgGl was achieved by substituting the amino acid residue Asn (N) with His (H) (i.e., N298H).

[0310] The amino acid numbers / positions discussed in this specification are based on sequences included in the sequence listing, which is part of this specification. It should be noted that the position discussed above at the 298th amino acid glycation point corresponds to the 297th amino acid glycation point in the native IgG1 molecule, which is numbered according to the European numbering system used for IgG1. Therefore, the 298th amino acid of this application corresponds to the 297th amino acid glycation point in U.S. Patent No. 7,501,494.

[0311] Fv domain deimmunization of mAb dB4 heavy chain ( FIG. 4 ) and light chains ( FIG. 5 The CDR1, 2, and 3 regions of MAb dB4 contain the amino acid sequences of SEQ ID NOs: 1 to 6, respectively (Table 4). The complete amino acid sequences of the MAb dB4 heavy and light chains are shown below. FIG. 4 and 5 As shown in SEQ ID NOs: 7 and 8.

[0312] It was found that mutations located at certain amino acids in the antibody heavy chain improved (extended) the half-life of mAb dB4. Specifically, the half-life of the humanized antibody was improved when the Fc amino acids at positions 253 (Met), 255 (Ser), and 257 (Thr) of the heavy chain were replaced with Tyr, Thr, and Glu, respectively. The full-length sequence of the modified heavy chain of the humanized mAb dB4 antibody is shown below. FIG. 6 As shown in SEQ ID NO: 9, the modified humanized antibody mAb dB4 comprises a light chain having the amino acid sequence of SEQ ID NO: 8 and a heavy chain having the amino acid sequence of SEQ ID NO: 9.

[0313] The unique sequence and structure of mouse mAb B4 and humanized mAb dB4 feature a glycosylation residue, Asn, located at amino acid position 101 (Asn101) in the heavy chain of the Fv region. This glycosylation site is unique to its Fv region location; it is unexpectedly hidden within the Fv domain and can only be exposed for enzymatic cleavage through denaturation of the entire antibody molecule. Initially, the presence of this glycosylation in the Fv region complicates antibody properties. However, modifications to the glycan chain or binding site can impair the antibody's affinity for CD4. Therefore, this unique N-glycosylation site in the Fv region is crucial for antibody binding to CD4.

[0314] FIG. 7 The complete length of the heavy chain amino acid sequence of mAb dB4 is specified, emphasizing... FIG. 4 and 6 At the glycosylation, substitution, and mutation sites discussed above.

[0315] Example 6. Demonstration of biological equivalence between mAb dB4 and its parental mAb B4 using MT-2 microplaquing, PBMC neutralization assays and PhenoSense entry assays

[0316] Extensive comparative studies were performed to assess the biological equivalence of the deimmunized / humanized, Fc-glycosylation-free mAb dB4 and the parental murine mAb B4 to ensure that the humanized version could be used for further toxicity / safety and efficacy studies in primates and humans. The results from these comparative studies are summarized below.

[0317] (1) High sensitivity HIV-1 neutralization assays were performed in MT-2 microplaquing and mitogen-stimulated PBMC assays using representative HIV isolates from clades A, B, C, D and E, and from clades C and E, respectively. The humanization of the murine mAb B4 to form mAb dB4 using deimmunization technology did not result in a loss of HIV neutralization activity. The comparative results are shown in the MT-2 microplaquing (Table 5) and PBMC-based assays (Table 6).

[0318] (2) Because the IC 50 s and IC 90 s measured for all HIV isolates from all clades were within two-fold of each other, the biological equivalence between the murine mAb B4 and mAb dB4 was demonstrated.

[0319] (3) HIV entry inhibition by mAb B4, mAb dB4 and two other known monoclonal neutralizing antibodies against HIV-env (2F5 and 2G12 HIV) was evaluated on the cell line U87 expressing dual receptors (CXCR4 and CCR5 or X4 / R5) and selected HIV isolates of various tropism (including JRCSF (R5), HXB2 (X4), 92TH594 (R5 / X4), primary isolate #5 (R5), primary isolate #6 (R5) and primary isolate #7 (R5)). In this assay, HIV entry inhibition was evaluated by Monogram Biosciences using a pseudotyped virus (pseudotyped virus carrying the envelope glycoprotein from any of hundreds of HIV strains and a luciferase). Quantification of bioluminescence produced in U87-CD4+ / CCR5+ / CXCR4+ cells, which were bioengineered to express luciferase under the control of HIV tat, was used to determine the neutralization effect of the antibodies. The results of this assay are shown in Table 7, which demonstrates that:

[0320] a. When compared with the two most potent anti-HIV Env antibodies, 2F5 (column 1) and 2G12 (column 2), murine mAb B4 (MuB4; column 4) was more potent in inhibiting HIV entry than the two. Corresponding IC50 values ​​for different HIV isolates. 50 The values ​​of s are 0.04 vs 4 and 0.8; 0.4 vs 0.07 and 0.5; 0.05 vs 3 and 1.3; 0.05 vs 50 and 20; 0.05 vs 2 and 3; 0.03 vs >100 and 2; and

[0321] b. When tested with the same representative HIV isolates of various tropisms, mouse mAb B4 (MuB4; column 4) and mAb dB4 (column 3) were detected at their IC50 values. 50 Surprisingly, s are equal.

[0322] Therefore, it is fully demonstrated that mouse mAb B4 and deimmunized mAb dB4 (both have the same CDRs in the heavy and light chains) are bioequivalent, and the functional characteristics of the two antibodies are representative of each other in different in vitro and in vivo experiments.

[0323] Example 7. (A) dB4 and mAb B4 binding activity against HPB-ALL cells; (B) dB4 binding activity against PBMC CD4-positive cells; (C) dB4 binding activity against recombinant CD4; and (D) characteristics of dB4 and gp120 binding activity against HPB-ALL cells.

[0324] 1. Background

[0325] Although the qualitative aspects of antigen binding and functional properties are known, as demonstrated by neutralization assays for mAbdB4 and its parent mouse mAb B4 as previously illustrated, cell binding curves for mAb B4 and mAb dB4 on CD4+ T lymphocytes have not been previously studied.

[0326] Cell binding curves of murine mAb B4 and its humanized, Fc-deglycosylated IgG1 monoclonal antibody mAb dB4 were tested in normal human blood CD4+ T lymphocytes and in CD4+ T-leukemia HPB-ALL cells. HPB-ALL cells were used because mAb B4 was selected by immunizing mice with HPB-ALL cells, as discussed in Example 1. Routine cell binding was assessed using flow cytometry (FACS) analysis, and results were denoted as EC based on mean fluorescence intensity (MFI). 50 or IC 50Values. In addition to assessing the general cell binding of the antibodies, the absolute binding affinity (Kd) and maximum binding capacity (Bmax) of the native dB4 IgGl molecule to HPB-ALL cells were also investigated.

[0327] 2. Materials

[0328] 2.1. Culture medium and reagents

[0329] RPMI-1640 medium and fetal bovine serum for culturing HPB-ALL cells were from Gibco (cat. nos. 11875-093 and 10091-148, respectively). Bovine serum albumin (BSA) was from ApplicChem (cat. no. A-0850). Culturing of cells with test antibodies was performed in V-bottom 96-well plates from NUNC (cat. no. 249662). Microdilution tubes (1.2 mL) for sample preparation were from Bertec (cat. no. 1710-00). Cells were fixed with 2% formaldehyde; samples were diluted in PBS (pH 7.4) containing 0.05% BSA and 0.05% sodium azide; and wash buffer was PBS (pH 7.4) containing 0.05% sodium azide.

[0330] Binding of mouse mAb B4 and humanized mAb dB4 was followed using goat anti-mouse IgG-FITC (Sigma, cat. no. F8264) and goat F(ab')2 anti-human IgG Fc gamma-FITC (Jackson ImmunoResearch, cat. no. 109-096-098), respectively. The dB4-Alexa 488 conjugate (throughout the article referred to by the abbreviation "dB4-Alexa") was manufactured in-house at UBI (UBI lot no. 0102143). The B4-biotin conjugate was from UBI (lot no. 051807). Sheep anti-hIgG-HRP was from The Binding Site (cat. no. AP004); Extravidin-HRP was from Sigma Aldrich (cat. no. E2886); and soluble rCD4 was from R & D System (cat. no. 514-CD-050). Peptide p2704a HIV envelope was from UBI, recombinant gpl20 MN was from ImmunoDiagnostics (cat. no. 1021-2). Blood CD4 +T cells were tracked for conjugation. Fluorescent beads from LinearFlow™ Green Flow Cytometry Intensity Calibration Kits (Molecular Probe) were used as a reference standard to quantitate the relative fluorescence of labeled cells. Other fluorescent detectors used were: FITC-ChromPure Goat IgG, F(ab')2 fragment (Jackson ImmunoResearch, cat# 005-090-006); CD3 PE (ASR) (BD Biosciences, cat# 340662); CD45 PerCP (ASR) (BD Biosciences, cat# 340665).

[0331] 2.2. HPB-ALL cells and peripheral blood CD4 + T cells

[0332] The HPB-ALL cell line (human thymus-derived acute lymphoblastic leukemia cell line) was provided by DSMZ ACC. PBMC CD4 + T cells (freshly drawn from healthy blood donors into EDTA-vacutainers) were derived from peripheral blood leukocytes (PBL) after lysis of red blood cells with a hypotonic solution containing ammonium chloride (8.3 g / L ammonium chloride, 0.84 g / L sodium bicarbonate, and 29.4 mg / L EDTA in pH 7.4).

[0333] 2.3. Murine mAb B4 and humanized dB4 mAb

[0334] The murine monoclonal B4 IgGl (UBI lot# 120197) was provided by a hybridoma procedure using HPB-ALL cells as immunogen. The B4-derived humanized dB4 IgGl was made Fc-glycosylation free by N298H (Example 5).

[0335] 2.4. ELISA and FACS detection

[0336] 96-well plates were from Nalge NUNC International, flat bottom for optical assays (Cat. 442404) and V-bottom for cell culture (Cat. 249570). Optical density was read on a VersaMax microplate reader (Molecular Devices). Fluorescent dyes were detected with a BD FACSCalibur scanner (DB Biosciences); and data were obtained with the associated Cell Quest software. Binding data from ELISA and FACS were imported into SigmaPlot 11 software for quantitative analysis.

[0337] 3. Method

[0338] 3.1. Binding of dB4 to HPB-ALL cells

[0339] 3.1.1. Equilibration time experiment

[0340] On V-bottom microplates, 0.1 mL per well of a solution containing 2 x 105cells was added, centrifuged, and the liquid discarded. Cells were incubated on ice with 100 μL of dB4 solutions at various concentrations up to 100 ng / mL for various durations up to 180 minutes. Supernatants were collected at the incubation time to determine the free, unbound antibody drug. The bound fraction was calculated using the total drug concentration added minus the free fraction. 5

[0341] Free dB4 concentration in the bound solution was quantified using an ELISA. Briefly, this assay involved the use of a sheep anti-hIgG L (0.5 μg / mL) cocktail coated on NUNC Maxisorp microplates, and a sheep anti-huIgG-HRP (1 / 1000 dilution) as the detection protein. Free dB4 concentration in unknown samples was determined based on a calibration standard in the range of 0.14-18.5 ng / mL.

[0342] 3.1.2. Direct binding experiment with dB4

[0343] On V-bottom microplates, 0.1 mL per well of a solution containing 2 x 105cells was added, centrifuged, and the liquid discarded. Cells were incubated on ice with 100 μL of dB4 solutions at various concentrations up to 100 ng / mL for various durations up to 180 minutes. Supernatants were collected at the incubation time to determine the free, unbound antibody drug. The bound fraction was calculated using the total drug concentration added minus the free fraction. 5 ​Solutions of 4 x 105cells were centrifuged and the liquid discarded. The cells were incubated with 100 μL of dB4 solution at various concentrations up to 2000 ng / mL for 1 hour on ice. After incubation, the dB4 was removed and a fresh solution of dB4 at the same concentration as used for the initial incubation was added to the cells and the cells were incubated for an additional 1 hour on ice. This step was repeated once. Cells from three incubations (passages) were studied. After the third incubation, the cells were washed once at 300 g for 5 minutes and stained with 100 μL of goat F(ab)2 anti-huIgG Fc-FITC (250 ng / mL) for 30 minutes on ice. The cells were washed once and the liquid discarded after centrifugation. 200 μL of binding solution was added to each well and transferred to a microdilution tube for flow cytometric analysis. The binding intensity (mean fluorescence intensity, MFI) was determined on a FACS based on an injection of 5,000 cells per sample.

[0344] 3.1.3. Binding affinity assay for dB4

[0345] In a centrifuge tube, dB4 antibody was added to HPB-ALL cells (0.5 mL) at a concentration of 3.1-2000 ng / mL (0.5 mL) and incubated for 1 hour on ice with gentle shaking. The absolute binding affinity at saturation binding was determined by quantifying the free dB4 concentration ([F]) in solution by ELISA and calculating the bound fraction ([B]) as described above for the equilibrium assay. The resulting saturation free-vs-bound concentration curve was analyzed by curve fitting on SigmaPlot based on the equation [B] = Bmax■{[F] / ([F] + Kd)}, where [B] and [F] represent the bound and free concentrations, respectively. 5 In a centrifuge tube, dB4 antibody was added to HPB-ALL cells (0.5 mL) at a concentration of 3.1-2000 ng / mL (0.5 mL) and incubated for 1 hour on ice with gentle shaking. The absolute binding affinity at saturation binding was determined by quantifying the free dB4 concentration ([F]) in solution by ELISA and calculating the bound fraction ([B]) as described above for the equilibrium assay. The resulting saturation free-vs-bound concentration curve was analyzed by curve fitting on SigmaPlot based on the equation [B] = Bmax■{[F] / ([F] + Kd)}, where [B] and [F] represent the bound and free concentrations, respectively.

[0346] 3.1.4. Competition of dB4 and B4 with B4-biotin

[0347] On flat-bottom microplates coated with a mixture of sCD4 (0.5 μg / mL) and p2704a peptide (2.0 μg / mL), 0.1 mL of dB4 or B4 solution (concentration 0.78-100 μg / mL) was added in the presence of B4-biotin (10 μg / mL) and incubated for 1 hour at room temperature. The following competition binding to the captured mixture was detected by Extravidin-HRP for bound B4-biotin and determined by an ELISA reader.

[0348] 3.1.5. Competition of dB4 and B4 with dB4-Alexa

[0349] On V-bottom microplates, 0.1 mL of dB4-Alexa (concentration 0.78-100 μg / mL) was added per well in the presence of B4-biotin (10 μg / mL) and incubated for 1 hour at room temperature. The following competition binding to the captured mixture was detected by Extravidin-HRP for bound B4-biotin and determined by an ELISA reader.5 Cells were cultured in a solution of 100 µL of dB4 or B4 solution (concentrations up to 2000 ng / mL) in the presence of dB4-Alexa (250 ng / mL) on ice for 1 hour. Cells were washed once and the liquid was discarded after centrifugation. 200 μL of binding solution was added to each well and transferred to a microdilution tube for flow cytometry analysis. Binding strength (mean fluorescence intensity, MFI) was determined on FACS based on an injection of 5,000 cells per sample.

[0350] 3.1.6. Competition between dB4C7 and gp120 MN and dB4C7-Alexa

[0351] Add 0.1 mL of solution containing 2 x 10⁻⁶ ppm to each well of the V-bottom microplate. 5 Cells were centrifuged and the liquid was discarded. Cells were incubated on ice for 1 hour in 100 µL of dB4C7 or gp120 MN solution (concentrations up to 200 nM, ~30 µg / mL) at dB4-Alexa (250 ng / mL). Cells were washed once and the liquid was discarded after centrifugation. 200 μL of binding solution was added to each well and transferred to a microdilution tube for flow cytometry analysis. Binding strength (mean fluorescence intensity, MFI) was determined on FACS based on an injection of 5,000 cells per sample.

[0352] 3.2. Binding of dB4 to blood CD4+ T lymphocytes

[0353] 3.2.1. Temperature dependence of dB4 combined with test

[0354] To simulate the physiological environment, dB4 (UB-421) binds to (covers or occupies) CD4 after intravenous injection. + CD4 receptors on T cells were cultured at 37°C with a freshly collected EDTA-blood solution and an equal volume of dB4 in dilution buffer (dB4 concentration up to 100 µg / mL). For comparison, other sample groups were simultaneously cultured on ice. After 1 hour of culture, the samples were lysed at room temperature for 10 minutes with 20 times the volume of erythrocyte lysis buffer to produce peripheral blood leukocyte (PBL) fractions.

[0355] PBL fractions were centrifuged, washed, and then resuspended in an equal volume of PBS buffer containing 1.0% BSA and sodium azide (e.g., 0.1 mL blood and 0.1 mL dilution buffer). PBL samples were stained with 0.1 mL of a mixture of goat F(ab)2 anti-hlgG Fc-FITC, anti-CD3-PE, and anti-CD45 perCP on ice for 30 minutes. After washing, samples were fixed with 2% formaldehyde and subjected to FACS analysis based on a 10,000 cell injection. The T lymphocyte population was selected using anti-CD3-PE.

[0356] 3.2.2. Direct binding

[0357] The direct binding of dB4 to blood CD4+ T cells was analyzed in three male and female subjects, where fresh drawn EDTA-blood was incubated with dB4 for 1 hour at 37°C. MAb dB4 bound to CD4+ T cells and an apparent saturation was found to be reached at a concentration range of 0.2-200 ng / mL. The T lymphocyte population was selected using anti-CD3-PE. This experimental procedure was identical to the one described above for the temperature dependent binding experiment, where the cells were stained with goat F(ab)2 anti-hlgG Fcγ-FITC to follow the binding of dB4.

[0358] 3.2.3. Unoccupied binding sites

[0359] Similar to the direct binding experiment described above, the extent of unoccupied binding sites left behind after the binding of dB4 was investigated in each of three male and three female individuals at the same occasion (before complete receptor occupation). This experimental procedure was identical to the one described above for the direct binding experiment, except that it used a fixed amount of dB4-Alexa (concentration 250 ng / mL) to visualize the extent of unoccupied, free binding sites.

[0360] 3.2.4. Calibration beads

[0361] To investigate both direct binding and unoccupied binding sites simultaneously, a combination of two Molecular Probes LinearFlow™ Kits (Cat. Nos. L14821 and L14823) was used to generate log(MFI)-vs-log(bead%) standard curves in each of six different occasions. This kit combination provides a broad calibration range of high and low intensity standards in flow cytometry experiments. As a reference standard, these fluorescent beads were used to quantify the relative fluorescence on cells labeled by either goat F(ab)2 anti-hlgG FcR-FITC or dB4-Alexa.

[0362] 4. Results and discussion

[0363] 4.1. Binding curve of dB4 and determination of its absolute binding affinity (Kd) to CD4-positive HPB-ALL cells

[0364] 4.1.1. Equilibration of binding activity to HPB-ALL cells

[0365] Before the complete characterization of the ligand-receptor binding reaction, the length of time to reach equilibrium for different concentrations of ligand, i.e. plateau, was defined, at which the rate of association equals the rate of dissociation. It is generally known that lower concentrations require longer times to reach equilibrium.

[0366] In the case of the dB4-CD4 interaction of CD4-positive HPB-ALL cells incubated on ice, about 60 min were required for the 2.0 ng / mL concentration and about 15 min for the 50 ng / mL concentration to reach a plateau in the number of % bound. A plateau was observed almost instantaneously for dB4 concentrations of 100 ng / mL (0.1 μg / mL) and higher.

[0367] These results indicate that the dB4 binding reaction can be achieved within 1 h for a wide range of concentrations (e.g. concentrations > 2.0 ng / mL). The binding assay can be performed cold and / or in the presence of 0.05% azide to avoid possible endocytosis of the ligand-receptor complex. Incubation at room temperature or at 37°C can lead to a faster plateau of the reaction. In the case of the above-mentioned progress, HPB-ALL cells, at three different cell passages, were incubated with dB4 on ice for 1 h. The supernatant was collected for ELISA determination of the free drug concentration and by subtraction from the total to provide the bound fraction. The absolute binding affinity and the binding capacity were calculated using the binding curve as shown in FIG. 8

[0368] 4.1.2. Direct binding to HPB-ALL cells

[0369] 2 x 10 5 HPB-ALL cells were incubated with dB4 (up to ~ 2000 ng / mL) on ice for 1 h, and the antibody exhibited a 4-parameter logit function of saturation binding curves, at which the extent of binding was measured using goat F(ab)2 anti-huIgG Fc-FITC and expressed as mean fluorescence intensity (MFI). Binding was close to saturation at concentrations of 200 ng / mL (0.2 μg / mL) and above. The average binding EC 50 ​Measured as 42.2 ng / mL (Table 8) with small between-passage variation (n=3). Absolute MFI values were also normalized to % MFI for between-passage comparison. This minimized standard deviation and EC 50 values remained essentially unchanged; the overall average binding EC 50 values measured as 42.9 ng / mL (Table 8).

[0370] 4.1.3. Binding affinity (Kd) and binding capacity (Bmax) to HPB-ALL cells

[0371] Supernatants after incubation (incubated 1 hour on ice) were collected for free (unbound) dB4 concentration [F] determination using ELISA, and thus the absolute binding affinity to dB4 was assessed using free drug vs. bound drug data plots as shown in Table 9. The average dissociation constant (Kd) was measured as 5.6 x 10 -11 M (range: 3.1 to 8.1 x 10 -11 M), and the maximum binding capacity (Bmax) was measured as 1.2 x 10 6 Ab (range: 0.93 to 1.4 x 10 6 These results indicate that dB4 binds to CD4 receptors on HPB-ALL cells with exceptionally high affinity, and that HPB-ALL cells have a high density, with over one million binding sites per cell for dB4 (Table 9). The CD4 receptor density on HPB-ALL cells is at least 20-fold higher than on blood CD4+ T lymphocytes, which have about 3.2 - 6.1 x 10 4 binding sites per cell.

[0372] 4.2. Comparison of dB4 and B4 binding to HPB-ALL cells

[0373] The challenge was to determine whether humanization of the deimmunized approach to make the murine B4 antibody dB4 altered the binding affinity, so this challenge was thoroughly investigated in two technical reports using a competition design.

[0374] First, the binding affinity of mAb B4 and mAb dB4 was investigated on ELISA microplates coated with a capture mixture of soluble CD4 (sCD4) and p2704a peptide. The p2704a peptide mimics the CD4-CCR5 receptor complex because it contains an antigenic epitope sequence on CCR5, which HIV-1 binds to for entry into CD4 cells. ELISA analysis showed that binding of B4-biotin to the coated sCD4 / p2704a mixture was inhibited in the presence of various concentrations of mAb B4 or mAb dB4. When mAb B4 or mAb dB4 antibodies coexisted and competed with B4-biotin for binding to the capture protein mixture, binding of B4-biotin was inhibited, and the IC50 values ​​for B4 and dB4 were [not specified in the original text]. 50 The values ​​were 5539 ng / mL and 8191 ng / mL, respectively. FIG. 9 IC of B4 to dB4 50 The ratio was 0.68, indicating that the humanized dB4 had a binding affinity equivalent to that of the mouse B4 antibody.

[0375] Furthermore, relative binding affinity was investigated using CD4-positive HPB-ALL cells, where B4 or dB4 antibodies coexisted and competed with dB4-Alexa for binding to the cellular CD4 receptor. FACS analysis showed that dB4-Alexa binding was inhibited, and the IC50 between B4 and dB4 was significantly lower. 50 The values ​​were 135 and 197 ng / mL, respectively. FIG. 10 IC of B4 to dB4 50 The ratio was 0.69, which is substantially the same as demonstrated by the ELISA example, further showing that humanized dB4 binds to the CD4 receptor with an affinity equivalent to that of mouse B4 antibody.

[0376] A comparative competitive binding inhibition assay, using the parental antibody B4 and its humanized antibody dB4C7 (UB-421), provided antibody binding profiles against CD4-positive T cells (HPB-ALL), as measured by mean fluorescence intensity (MFI) for a range of antibody concentrations (from 100 to 104 ng / mL). This assay further validated the data presented in Tables 5 and 6, demonstrating that the individual neutralizing activities of these two antibodies are comparable in the MT2 and PBMC assay systems.

[0377] When compared with its parent murine antibody B4, the results of these comparative studies indicate that humanization using deimmunization techniques did not significantly reduce the binding affinity of dB4C7 (UB-421) to the CD4 receptor.

[0378] 4.3. Binding characteristics of MAb dB4 to CD4

[0379] Evaluation of the binding characteristics of mAb dB4 to CD4.

[0380] 4.3.1. Binding of mAb dB4 to soluble CD4 and to cell-bound CD4

[0381] As discussed above, dB4 inhibited the binding of B4-biotin to sCD4 / p2704a with an IC 50 of 8191 ng / mL ( FIG. 9 ), and dB4 inhibited the binding of dB4-Alexa to HPB-ALL cells with an IC 50 of 197 ng / mL ( FIG. 10 ). These data interestingly demonstrate that dB4 has a higher binding affinity for CD4-positive T cells than for soluble CD4 (sCD4). Specifically, comparison of the IC 50 values of these two assays shows that dB4 has a binding affinity for CD4-positive T cells that is more than 40-fold higher than for soluble CD4.

[0382] 4.3.2. Comparison of dB4 and gpl20 MN binding to CD4 on HPB-ALL cells

[0383] A competition assay was performed to compare the binding affinities of dB4 and HIV gpl20 MN for CD4 bound to CD4-positive T cells. Specifically, the ability of dB4 and gpl20 MN to inhibit the binding of dB4-Alexa to CD4 on HPB-ALL cells was compared ( FIG. 11 ). In the first assay, dB4 inhibited the binding of dB4-Alexa to CD4 on HPB-ALL cells with an IC 50 of 1.8 nM. In the comparative assay, gpl20 MN inhibited the binding of dB4-Alexa to CD4 on HPB-ALL cells with an IC 50 of 97.2 nM. In light of these results, it was found that dB4 has substantially higher binding affinity for CD4 on HPB-ALL T cells than does gpl20 MN. Specifically, comparison of the IC 50 values of these two assays shows that dB4 has a binding affinity for CD4 that is at least 50-fold higher than gpl20 MN.

[0384] 4.3.3. Comparison of the binding affinities of dB4 and gpl20 MN for CD4

[0385] As discussed above, mAb dB4 binds to CD4 with a Kdof about 5.6 x 10 -11M (Table 9). Others have previously found, through crystallographic studies, that HIV-1 gp120 binds near the CD4 molecule domain 1 with a Kd of about 5 x 10 -9 M (Myszka, D.G., et al., "Energetics of the HIV gp120-CD4 binding reaction" Proc Natl Acad Sci U S A. Aug 1, 2000; 97(16): 9026-9031). Thus, comparison of the Kd values for dB4 and gp120 shows that the binding affinity of dB4 for CD4 is about 100-fold higher than that of gp120. This result is consistent with the findings above (based on IC 50 values, dB4 binds HPB-ALL cells at least 50-fold stronger than gp120 MN.

[0386] The in vitro results collectively show that the above discussion demonstrates that dB4 is sufficient to block or reduce HIV-1 infection by avoiding or shutting down HIV viral entry.

[0387] 4.4. Temperature-dependent binding of dB4 (UB-421) to blood CD4+ T cells

[0388] The binding of dB4 to CD4+ T cells in human blood at normal body temperature (37°C) was investigated to determine whether dB4 can be effectively administered to human subjects as a therapeutic drug. To mimic the physiological environment of the human body, dB4 C7 (UB-421) was incubated with freshly drawn blood at 37°C for 1 h, and a peripheral blood leukocyte (PBL) sample was provided using a red blood cell lysis step. Incubation on ice (4°C) was performed simultaneously. The PBL fraction was then stained with goat F(ab)2 anti-huIgG-FITC to visualize the direct binding of dB4 to CD4 receptors on CD4-selected T cells.

[0389] Compared to incubation at 4°C (data not shown), incubation of dB4 at 37°C for 1 h did not cause endocytosis of the ligand-receptor, as no change in MFI was observed for the mouse anti-CD4 (D2)-FITC. Mouse anti-CD4 (D2)-FITC (used for CD4+ T cell selection, at 50-1000 ng / mL) and dB4 (targeting the D1 domain of the CD4 receptor, at up to 200 ng / mL) did not compete for binding.

[0390] As shown for a blood sample from a female individual (Figure 4), the MFI of dB4 C7 (UB-421) was about 2-fold higher than that of mouse anti-CD4 (D2)-FITC. The MFI of dB4 C7 (UB-421) was about 2-fold higher than that of mouse anti-CD4 (D2)-FITC. The MFI of dB4 C7 (UB-421) was about 2-fold higher than that of mouse anti-CD4 (D2)-FITC. FIG. 12), peripheral blood mononuclear cells (PBMC) were incubated with dB4 for 1 hour at two different temperatures (37°C or 4°C). Selected CD4+ T cells were stained for observation of dB4 binding on FACS. FIG. 12 It was shown that dB4 binds to CD4 receptor with 5-fold higher affinity at 37°C than at 4°C, based on IC 50 Values. As expected, dB4 can reach the same maximum binding MFI at both temperature conditions. MFI values reflect the degree of receptor occupancy, especially when these values are normalized and expressed as % MFI.

[0391] 4.4.1. Direct binding of dB4 to blood CD4+ T cells

[0392] Binding curves of dB4 to CD4+ T cells in human blood at normal body temperature (37°C) were investigated. Freshly drawn blood from six adults (three males and three females) was used to evaluate the direct binding activity of dB4 to blood CD4+ cells at six different occasions (Table 10). After 1 hour incubation, peripheral white blood cells were isolated, stained with goat F(ab)2 anti-huIgG Fc-FITC, and dB4-bound CD4-selected T cells were analyzed by FACS. Samples were corrected with reference beads for fluorescence reading. The binding (EC 50 ) values were found to be between 2.6 ng / mL and 5.7 ng / mL, with an average EC 50 of 4.1 ng / mL. Likewise, the maximum %MFI values were between 68% and 93%, with an average of 77.8% (Table 10). These results reflect, respectively, inter-subject differences of little significance on binding affinity and receptor density.

[0393] 4.4.2. Unoccupied CD4 binding sites after receptor occupation with mAb dB4

[0394] Unoccupied, free CD4 binding sites on CD4+ cells were evaluated with dB4-Alexa, in conjunction with direct binding of dB4 to blood CD4+ cells at 4°C, as detected with goat anti-hIgG.

[0395] In the absence of dB4, the dB4-Alexa conjugate alone can approach ~100% of its maximum binding at a concentration of ~250 ng / mL FIG. 13At concentrations above 500 ng / mL, the dB4-Alexa conjugate can displace approximately 10% or more of the bound dB4 (data not shown). Specifically, when the receptor is occupied by dB4 at a near-saturation level of 250 ng / mL, the binding of dB4-Alexa (at 250 ng / mL) to the CD4 receptor is completely blocked.

[0396] As the binding of dB4-Alexa increased, receptor occupancy decreased with decreasing dB4 presence. Receptor occupancy and the degree of unoccupied binding sites were synchronized symmetrically, and the two curves intersected at a concentration of approximately 4.0 ng / mL. FIG. 13 This is combined with EC 50 The values ​​are consistent (Table 10).

[0397] Therefore, the overall results indicate that the use of 250 ng / mL dB4-Alexa and MFI, as well as %MFI, in vitro can serve as a suitable example for studying in vivo receptor occupancy after human subjects are administered dB4C7 (UB-421).

[0398] 5. Conclusion

[0399] (1) MAb dB4 interacts with the CD4 receptor located on HPB-ALL cells, exhibiting uniquely high activity and reaching immediate equilibrium on ice at concentrations above 50 ng / mL. The absolute binding affinity (Kd) was determined to be 5.6 x 10⁻⁶. -11 M, and at its highest value 1.2 x 10 6 Each dB4 molecule (Bmax) can bind to a single HPB-ALL cell, which has at least 20 times higher receptor density than normal blood CD4+ T cells.

[0400] (2) Humanized mouse B4 as dB4 mAb did not significantly alter its dB4 binding affinity to the CD4 receptor. Using a binding inhibition design, molecular-based ELISA (coated with sCD4 and the peptide p2704a containing the CCR5 antigenic epitope) and cell-based FACS on HPB-ALL cells, both antibodies equally blocked the binding of tracers (B4-biotin or dB4-Alexa). In these two experiments, the IC50 between B4- and dB4- was observed. 50 The ratio is approximately 0.7.

[0401] (3) The dB4 antibody binds to the CD4 receptor with an affinity at least 50 times greater than that of the HIV-1 envelope protein gp120 MN. Specifically, binding inhibition assays on HPB-ALL cells demonstrated that dB4 and gp120 MN have IC50 values ​​of 1.8 and 97.2 nM, respectively. 50Numerical inhibition of binding to the tracer dB4-Alexa. This result is also consistent with the high binding affinity (Kd) of dB4 (which was previously reported to be about 100 times higher than that of recombinant gp120).

[0402] (4) MAb dB4 binds to blood CD4+ T cells with a binding affinity similar to that for HPB-ALL cells. At low temperatures (4°C), the EC50 of dB4... 50 It is approximately 23.0 ng / mL. Furthermore, at normal body temperature (37°C), dB4 binds to blood CD4+ T cells with approximately 5 times the affinity, and binds to EC2+. 50 It was 4.8 ng / mL.

[0403] (5) Tests using blood samples from six human subjects demonstrated a direct (inverse) correlation between dB4 concentration and CD4 receptor occupancy. For example... FIG. 13 The opposite curves shown intersect at approximately 4.0 ng / mL, which corresponds to the average EC50 of dB4 for CD4. 50 The combined numerical values ​​are consistent. These overall results indicate that the in vitro determination of dB4-Alexa (concentration 250 ng / mL) and %MFI corrected with fluorescent beads can serve as an appropriate paradigm for studying receptor occupancy in human subjects after administration of dB4C7 (UB-421).

[0404] Example 8. Antibody B4 effectively inhibits cell-free and cell-to-cell transmission of HIV.

[0405] HIV particles typically spread throughout the body via cell-free transmission, where the virus spreads in the bloodstream and local environment to infect cells. The virus also possesses the ability to directly transfer from infected to uninfected cells through a mechanism requiring close cell-to-cell contact. This transmission occurs when an infected cell and an uninfected cell form a stable point of contact, directly transmitting HIV particles to the uninfected cell. Compared to cell-free transmission, cell-to-cell transmission is more efficient, faster, and does not require diffusion into the bloodstream.

[0406] Sigal, A., et al. reported in 2011 that the presence of the antiviral drug tenofovir drastically reduced infections originating from cell-free viruses, while infections involving intercellular transmission in co-culture assays showed significantly less susceptibility to the drug. FIG. 14). The reduction in sensitivity was sufficient to prevent the termination of multiple rounds of infection in the presence of the drug. The authors used a stochastic infection model to investigate replication from cell-to-cell spread in the presence of clinical drug concentrations and found that replication was intermittent, with no substantial accumulation of mutations. If cell-to-cell spread has the same properties in vivo, it can have deleterious consequences for the immune system, with dangerous factors to cause treatment failure in individuals and can contribute to viral persistence, thus, it is an obstacle to cure HIV infection.

[0407] Therefore, in order to assess its potential impact on treatment, it is important to assess the ability and efficacy of mAb B4 and mAb dB4 related antibodies to inhibit HIV cell-to-cell spread.

[0408] 1. Assay to measure antibody-mediated inhibition of HIV cell-to-cell spread

[0409] 1.1. Materials and methods

[0410] 1.1.1. Cells and viruses

[0411] Jurkat-inGLuc clones (from the National Institutes of Health AIDS Research and Reference Reagent Program) were selected, which have a reporter gene (luciferase) genetically inserted into the HIV-1 genome, as donor cells (because low expression of surface CD4 can minimize donor-vs-donor infection in co-culture assays with target primary CD4+ T cells). The reporter gene luciferase can be expressed in infected cells and serves as a marker of viral infection. These viral expression reporters in infected cells can be measured to quantify HIV-1 infection. Primary CD4 + T cells were used as target cells. Viruses UG266 and UG046 of clade D were used in this assay.

[0412] 1.1.2. Viral cell-to-cell spread assay

[0413] In this assay, donors were pre-incubated with serial dilutions of antibody B4 before mixing with the indicated HIV-1 strains and used a few days later (when ~10-75% of cells were Gag + positive). Then 1.5 × 10 6Final concentrations of 100 cells / ml were mixed in a 1 :2 ratio of donor and CD4-positive PBMC target cells in 96-well plates. After 48 hours, cells were stained for intracellular Gag and analyzed by flow cytometry. GLuc accumulated in the culture supernatant was detected using the BioLux Gaussia Luciferase Assay Kit (New England Biolabs) and a Berthold Technologies luminometer.

[0414] 1.1.3. IC 50 and IC 90 correction

[0415] Dose-response inhibition curves were plotted by fitting the data to a sigmoidal dose-response curve (variable slope). The percentage of inhibition was defined as (percentage of signal of untreated target cells - percentage of signal of antibody-treated cells) / (percentage of signal of untreated target cells) x 100. IC 50 and IC 90 were calculated accordingly.

[0416] 2. Results and discussion

[0417] Table 11 shows that antibody B4 can equivalently inhibit HIV (strains UG266 and UG046 of clade C) cell-to-cell and cell-free transmission when determined by the stringent 90% entry inhibition criterion. Specifically, fusion inhibition titers of 1 : 140 and 1 : 245 were found for the UG266 and UG046 strains, respectively, in cell-to-cell transmission assays, which correspond to neutralization titers of 1 : 136 and 1 : 234 in cell-free transmission neutralization assays. Higher fusion inhibition titers were observed for both strains in cell-to-cell transmission compared to cell-free transmission when determined by the 50% entry inhibition criterion.

[0418] These results demonstrate that antibody B4 has unique properties in its ability to inhibit HIV cell-to-cell and cell-free transmission when compared to all other neutralizing monoclonal antibodies against the HIV Env protein or other ART-drugs determined so far. These results show that only mAb B4 and mAb dB4 related antibodies qualify for the prevention of cell-free and cell-to-cell transmission of the HIV virus in individuals.

[0419] Example 9. Antibody UB-421 (dB4C7 or dB4) mediates reactivation of silent PBMCs for enhanced viral replication in HIV-infected individuals

[0420] 1. Background

[0421] HIV-1 infection of quiescent peripheral blood mononuclear cells (PBMCs) remains silent until subsequent cell activation. Using an in vitro model that mimics quiescent HIV-1 harbored in quiescent PBMCs by using cell culture conditions and methods that allow non-productive infection of quiescent T cells, the stimulatory effect of heat-inactivated HIV-1 (iHIV-1) or gpl20-anti-gpl20 immune complex on these quiescent PBMCs was investigated (Briant, L., et al., 1996).

[0422] This demonstrates that CD4 engagement of the envelope glycoprotein of heat-inactivated HIV-1 (iHIV-1) or gpl20-anti-gpl20 immune complex is sufficient to stimulate signaling pathways that control NF-κB (i.e., nuclear translocation) and AP-1 activation, which in turn involve the extracellular domain 1 (D1) and intracellular domains of CD4 with several kinases (Lck, Raf-1, MEK and ERK) to induce cell cycle progression, promote cell surface expression of the activation marker CD25, and stimulate proviral integration and virus production by the cells.

[0423] This was further confirmed by the independent scientific discovery of Than et al. (Than, et al., 1997) that cross-linking of CD4 molecules at the gpl20 binding site with an anti-CD4 monoclonal antibody induced latent infected PBMCs from HIV-infected patients to promote viral replication. The anti-CD4 mAb used in this experiment was Leu3a, which binds to the CDR2-loop of D1 of CD4. Specifically, Leu3 is directed against a linear epitope that represents a peptide with amino acids 47-64 within domain 1 of CD4 (Chiba, Y. 1992).

[0424] Furthermore, it was found that viral reactivation in quiescent PBMCs can be specifically induced by a monoclonal antibody directed against the CDR2-loop of domain 1 (D1) of CD4 and not by antibodies directed against other epitopes, such as the CDR3 of D1 or the region close to the D1 / D2 junction (Briant, L., et al., 1999) (see FIG. 15 , compare bars 4 with 5 and 6). This viral reactivation can be prevented by pre-adsorbing the CDR2-loop ligand with soluble CD4 (sCD4) (see FIG. 15 , compare bars 4 and 8).

[0425] It is therefore important to assess whether the antibody dB4C7 (UB-421), which has a high binding affinity for the region near CD4 domain 1, can mediate the reactivation of silent PBMCs for enhanced viral replication in HIV-infected individuals.

[0426] 2. Structure-activity of the B4 / dB4 binding site near Dl of CD4

[0427] 2.1. Competitive sequential binding inhibition of Leu3a binding to chimpanzee CD4-positive PBMCs by mAb B4

[0428] Chimpanzee PBMC cells isolated from two subjects (X282 and X301), mAb B4 (labeled with FITC) and Leu3a (labeled with PE) were used in this experiment. PBMCs were sequentially stained with individual antibodies and analyzed by cytofluorography. The data provided by this experiment are described in Table 12 and discussed below.

[0429] In single-label control samples, only cells stained with Leu3a were tested for Leu3a-PE binding positivity; only cells stained with mAb B4 were tested for B4-FITC binding positivity. Specifically, CD4+ cells (as detected by Leu3a) in uninfected chimpanzee samples (X282 and X301) were 25.5% and 44.0%, respectively, which were similar to those detected by mAb B4 (26.1% and 45.5%).

[0430] Exposure to mAb B4 after prior binding of Leu3a resulted in a number of double-stained (Leu3a+ / B4+) PBMC cells similar to single-label control cells stained with Leu3a or B4 alone (i.e., 24.5% and 46.7% for X282 and X301, respectively).

[0431] Conversely, exposure to Leu3a after prior binding of mAb B4 resulted in only mAb B4-stained PBMCs that were negative for Leu3a staining in single or double staining.

[0432] Overall, these results demonstrate a one-way inhibition by the antibody B4-FITC against Leu3a-PE. That is, pre- Leu3a binding failed to block B4 binding; however, pre-B4 binding could block Leu3a binding. The conclusion is that mAb B4 recognizes a conformational epitope encompassing the CDR2 region of CD4 domain 1 (which region is recognized by antibody Leu3a), and mAb B4 binds this region of CD4 with a higher affinity than antibody Leu3a. These data support this conclusion.

[0433] 2.2. Competition inhibition of B4 binding to rsCD4 by immune sera against HIV RC peptide (amino acids 39-66) (ELISA method)

[0434] The binding affinity of mAb B4 to intact recombinant soluble CD4 (rsCD4) was assessed by a competition inhibition assay using immune sera against the CDR2 region of CD4 domain 1.

[0435] 2.2.1. Anti-HIV RC polyclonal antibodies

[0436] Guinea pigs were immunized with a cyclic peptide comprising amino acids 39-66 of CD4 to generate polyclonal antibodies against the CDR2 region of CD4 domain 1. This cyclic peptide is referred to as the HIV receptor complex peptide (HIV RC peptide) in this assay, and is like the peptide p2240c described previously in Wang et al. 2002.

[0437] Specifically, week 0 was formulated in complete Freund's adjuvant, and weeks 3 and 6 were formulated in incomplete Freund's adjuvant, followed by monthly boosts formulated in incomplete Freund's adjuvant. Serum from guinea pigs against the HIV RC peptide was collected at specific time points after immunization of 4-6 week old Duncan Hartley guinea pigs with 100 μg in 0.5 ml per dose formulated in incomplete Freund's adjuvant.

[0438] The polyclonal antibodies provided are referred to as "anti-HIV RC polyclonal antibodies".

[0439] 2.2.2. Competition inhibition of B4 binding to rsCD4 by anti-HIV RC polyclonal antibodies

[0440] A 96-well microplate was coated with intact rsCD4 at a volume of 0.1 mL at a concentration of 0.08 pg / mL per well. Prior to binding with biotinylated B4-antibody, the guinea pig serum (collected at weeks 0, 3, 6, 9, 12, 14, 16, and 19 after immunization with an immunogen against HIV RC peptide (amino acids 39-66 of CD4)) was diluted at a ratio of 1 :30 and incubated with the wells before using the binding with avidin-HRP as a tracer. Negative control sera (RC homolog) collected from unimmunized guinea pigs at the same period of the entire experiment were also tested.

[0441] FIG. 16 It was shown that anti-HIV RC polyclonal antibodies provided at week 6 after the primary immunization could significantly inhibit the binding of biotinylated-B4 to rsCD4, and that anti-HIV RC polyclonal antibodies provided at week 9 after the primary immunization could achieve nearly complete inhibition.

[0442] This competitive binding inhibition experiment further demonstrated that the binding site of mAb B4 is located near the CDR2 loop of CD4 domain 1, although direct binding to this peptide by mAb B4 is not significantly due to the preferential binding of mAb B4 to the conformational structure of membrane-bound CD4.

[0443] 2.3. Reactivation of silent CD4-positive T cells for enhancing virus production in HIV-infected individuals upon cross-linking of mAb dB4

[0444] The ability of mAb dB4 to activate silent CD4+ cells was evaluated by treating the cells with mAb dB4 and observing TNF-a production, viral load, and cell proliferation.

[0445] In this experiment, human IgG was coated on 8-well culture plates by incubating the plates with 200 pL goat anti-human IgG (Jackson ImmunoResearch) at 37°C for 1 hour. The coated culture plates were kept in a 4°C refrigerator until further use in this experiment.

[0446] PBMCs from HIV patients were thawed for 1.5 hours according to standard procedures. The PBMCs were treated with mAb dB4 (experimental group), PMA+PHA (positive control group), or just culture medium (negative control group) as described below to evaluate the activation of silent CD4+ cells.

[0447] 2.3.1. MAb dB4 treatment

[0448] PBMCs were treated with mAb dB4 (at a concentration of 3 pg / 10 6Cells were treated with mAb dB4 for 1 hour at 4°C to initiate cross-linking of CD4 on the cells. Cells were treated with mAb dB4, followed by washing, and cultured for 7 days on coated 48-well plates in RPMI media with 10% fetal bovine serum. Uncoated wells were used as negative controls. Culture supernatants were frozen at day 0, day 2, and day 7 for later evaluation. Supernatants were removed from cells after 30 minutes of incubation at 4°C to provide time point day 0 as the mAb dB4 sample.

[0449] 2.3.2. PMA+PHA treatment

[0450] Cells were treated with 0.1 μΜ phytohaemagglutinin (PHA) plus 15 μg / mL phorbol myristate acetate (PMA) (Sigma) (PMA+PHA) for 7 days on coated 48-well plates in RPMI media with 10% fetal bovine serum as a positive control for reactivation of quiescent CD4+ cells. Uncoated wells were used as negative controls. Culture supernatants were frozen at day 0, day 2, and day 7 for later evaluation. Supernatants were removed from cells after 30 minutes of incubation at 4°C to provide time point day 0 as the PMA+PHA sample.

[0451] 2.3.3. Media only

[0452] Cells were cultured for 7 days on coated 48-well plates in RPMI media with 10% fetal bovine serum as negative controls (media only). Uncoated wells were used as additional negative controls. Culture supernatants were frozen at day 0, day 2, and day 7 for later evaluation. Supernatants were removed from cells after 30 minutes of incubation in media at 4°C to provide time point day 0 as the media only sample.

[0453] 2.3.4. Analysis of CD4+ reactivation

[0454] Reactivation of CD4+ cells was determined by assessing TNF-α production, viral load, and cell proliferation. Results from this experiment are summarized in Table 13.

[0455] Solutions from all samples were analyzed using standard methods, including: (1) TNF-α concentration was analyzed using quantitative ELISA; (2) HIV viral load was analyzed using RT PCR; (3) cell number; and (4) viability was analyzed using trypan blue.

[0456] Specifically, the data show that mAb dB4 cross-linked PBMC cells from HIV patients caused a moderate production of TNF-α compared to the negative control with media alone (below the limit of detection) and cells stimulated with PMA+PHA (about 3 to 5 times higher than cells coated with mAb dB4).

[0457] Similarly, the mAb dB4 samples proliferated at a similar rate as the negative control with media alone; however, the PMA+PHA stimulated cells had a greater degree of proliferation than cells cross-linked with mAb dB4 (5 times more cells in the PMA+PHA culture group than the mAb dB4 culture group at day 7).

[0458] However, the HIV viral load was significantly increased in cells cross-linked with mAb dB4 compared to the media control and PMA+PHA stimulated cells. Specifically, cells cross-linked with mAb dB4 had a 151% and 220% increase in viral load at days 2 and 7, respectively, when compared to the negative control with media alone; however, the PMA+PHA culture had the least amount of viral production (55% and 78% at days 2 and 7, respectively), despite having a 5 times greater increase in cell proliferation.

[0459] 3. CONCLUSIONS

[0460] (1) It was found that the murine mAb B4 recognizes a conformational site on CD4 that is close to the site recognized by the antibody Leu3a (amino acids 47-64 in the CDR2 region). Based on the comparative tests described in Example 7, it is expected that mAb dB4 has the same recognition characteristics as mAb B4 described herein.

[0461] (2) The binding of murine mAb B4 to intact rsCD4 was inhibited using a polyclonal antibody to a cyclic peptide (HIV RC peptide) containing amino acids 39-66 in the CDR2 region of domain 1 of CD4. These results show that mAb B4 recognizes amino acids 39-66 in CD4, which corresponds to the CDR2 loop of Dl of CD4. Based on the comparative tests described in Example 7, it is expected that mAb dB4 has the same recognition characteristics as mAb B4 described herein.

[0462] (3) It was found that cross-linking of CD4 with mAb dB4 to HIV infected PBMC CD4+ T cells activates viral production. Specifically, mAb dB4 induced TNF-α production and enhanced HIV production without inducing cell proliferation (as shown in Table 13).

[0463] (4) Based on the results provided by this example, mAb dB4 (including UB-421) can mediate the reactivation of silent PBMCs for enhanced virus production in HIV infected individuals.

[0464] Example 10. MAb dB4C7 and anti-HIV RC polyclonal antibodies inhibit antigen- induced T cell proliferation and cytokine (IL2 and IFN-g) production by CD4 positive T cells, thus disrupting the HIV pathogenic cycle of pyroptosis

[0465] 1. Background

[0466] Recent reports show that when HIV virus infects permissive, activated CD4+ T cells, cell death occurs silently through caspase-3 dependent apoptosis (Doitsh, G., et al., 2014). Conversely, when R5 or X4-tropic HIV fails to infect non-permissive, silent CD4+ T cells from lymphoid tissues, these cells die through caspase-1 dependent pyroptosis, a potent inflammatory form of programmed cell death. Interferon-inducible factor 16 (IFI16), considered a host DNA sensor, can recognize incomplete HIV reverse transcription products, triggering caspase-1 activation (Monroe, K.M., et al., 2013). In most human lymphoid tissues, including tonsils, lymph nodes and spleen, the subpopulation of activated and permissive cells represents 5% or less of the total number of CD4 T cells, while non-permissive silent cells represent 95% or more of the target for HIV. Thus, caspase-1 mediated pyroptosis appears to be mainly responsible for driving CD4 T cell death after HIV infection of these lymphoid tissues, rather than caspase-3 mediated apoptosis. Analysis of fresh lymph nodes from subjects infected with R5-tropic HIV further supports these findings, where caspase-1 and IL-1 β can be detected in the cortical area rich in silent CD4 T cells, while caspase-3 activity is found in anatomically distinct germinal centers where productive infected cells are found.

[0467] Pyroptosis can contribute to the rapid clearance of various bacterial infections by removing intracellular replicative niches and enhancing host defense responses through the release of proinflammatory cytokines and endogenous danger signals. However, in chronic inflammatory conditions that are pathogenic, such as HIV infection, pyroptosis is not a protective response and does not result in clearance of the primary infection. In fact, pyroptosis appears to create a vicious pathogenic cycle in which dying CD4 T cells release inflammatory signals to attract more cells into the infected lymphoid tissue, which in turn die and produce more inflammation. These events establish a chronic state of inflammation that stimulates disease progression and tissue damage. Chronic inflammation can also contribute to the maintenance of the latent HIV viral reservoir, which can stimulate the constant proliferation of memory CD4 T cells.

[0468] Depletion of CD4 T cells and development of chronic inflammation are characteristic processes in the pathogenic mechanisms of HIV that drive disease progression, and pyroptosis provides an unexpected link between these two disease-promoting processes.

[0469] The above information shows that mechanisms to inhibit CD4+ cell proliferation and / or inflammatory cytokine production initiated by antigen stimulation of CD4+ cells can reduce or diminish pyroptosis in lymphoid tissues that occurs during HIV infection.

[0470] 2. Experiments

[0471] Studies were conducted to determine whether mAb dB4 can inhibit the development of chronic inflammation in HIV-infected individuals to disrupt the pathogenic cycle caused by pyroptosis. Inhibition of cytokine production initiated by antigen stimulation can help to alleviate the burden of pyroptosis in many silent T cells (silent T cells that have failed HIV infection), thereby disrupting the HIV pathology that results from CD4-positive T cell depletion caused by cytokines.

[0472] An in vitro model using Staphylococcus aureus enterotoxin type B (SEB) was used to evaluate the ability of mAb dB4C7 (UB-421) to inhibit T cell proliferation of PBMCs in normal and HIV-infected individuals. SEB is a superantigen that has the ability to stimulate all T cells bearing a specific T cell antigen receptor (TCR) and can induce massive cytokine production.

[0473] Functional analysis of normal human donors (n=3) and HIV infected donors (n=6, patients not treated with ART, CD4+ number >200, viral load >10,000) were performed in collaboration with Drs. Huyen Cao and Mohamed Elrefaei to assess whether mAb dB4C7 (UB-421) or anti-HIV RC polyclonal antibodies directed against the CDR2 region of D1 of CD4 could inhibit cell proliferation and cytokine (IL2 and IFN-g) production.

[0474] 2.1. Study subjects and samples

[0475] HIV-positive volunteers not treated with ART (n=6) were recruited from the REACH cohort (San Francisco). Three age-matched HIV-seronegative control volunteers were also included in this study. PBMCs were isolated and cryopreserved in liquid nitrogen until the time of the study.

[0476] 2.2. Saturation concentration of mAb dB4C7 or purified anti-HIV RC polyclonal antibodies

[0477] CD4+ T lymphocytes were first stained with mAb dB4C7 IgG or anti-HIV RC polyclonal antibody IgG, respectively, in an indirect immunofluorescence assay, followed by staining with Alexa-goat anti-Hu IgG or Alexa-goat anti- guinea pig IgG. To detect the percentage of positive cells, the resulting stained cells were analyzed by flow cytometry. The titers of mAb dB4C7 and anti-HIV RC polyclonal antibodies were determined at 2-fold dilutions between 50 µg / mL and 0.0025 µg / mL. Antibody titration of mAb dB4 and anti-HIV RC antibodies was determined as % CD4 binding against antibody concentration (µg / mL). These antibody titers were evaluated before use in T cell functional tests in HIV infected and normal individuals.

[0478] FIG. 17 Saturation concentrations of each reagent used in the functional assay were 1 µg / mL for mAb dB4 (dB4C7) and 25 µg / mL for anti-HIV RC polyclonal antibodies.

[0479] 2.3. Proliferation of CD4+ or CD8+ T cells

[0480] Cell proliferation was analyzed using the carboxy-fluorescein succinimidyl ester (CFSE) fluorescent assay, which produces a decrease in carboxy-fluorescein diacetate, succinimidyl ester (CFDA-SE) staining upon cell division. CFSE was used as 3 H-thymidine (proliferation) test surrogate.

[0481] PBMCs were incubated with saturating concentrations of mAb dB4C7 or purified anti-HIV RC polyclonal antibodies to coat the CD4 receptors on the cell surface. Cells were also incubated with anti-HIV RC isotype (at a concentration of 25 μg / mL) and PHA (10 μg / ml; Sigma-Aldrich) as negative and positive controls, respectively.

[0482] PBMCs were labeled with CFDA-SE (Molecular Probes, Eugene, OR) in PBS, followed by fluorescence quenching with 100% FCS (Sigma-Aldrich, St. Louis, MO). After washing with PBS, cells were resuspended in RPMI 1640 (Sigma-Aldrich) with 10% FCS.

[0483] Cells were then incubated in the presence of SEB antigen (1 μg / mL) for 5 days at 37°C with 5% CO2, and analyzed for surface marker expression.

[0484] Flow cytometry was used to analyze CD4+ (PE, D2) and CD8+ (PercpCY5.5) cell populations within the CD3+ (Amcyan) cell population, and this population was further defined for % CFSE positive cells (as % proliferating cells). Forty thousand (40,000) lymphocytes per sample were acquired using an LSR II (BD Biosciences, Mountain View, CA), and analyzed using FLOWJO software (TreeStar, San Carlos, CA). Results were determined as the percentage of dividing CD4 (or CD8) T cells. All test participants demonstrated significant cell proliferation following PHA stimulation. Cell proliferation of CD4 T cells without SEB antigen stimulation was <0.5% (negative control).

[0485] 2.4. Intracellular staining assay to measure cytokine (IL2 and IFN-γ production)

[0486] PBMC (0.5 x 10 6 Cells were cultured with SEB antigen (1 µg / mL) at 37°C with 5% CO2 for 2 hours. Cells were washed with PBS (wash buffer) containing 0.1% FCS and then resuspended in lysis buffer (BD Biosciences) for 10 minutes at room temperature for fixation. Cells were washed once with wash buffer and then resuspended in 0.5 mL of membrane permeabilizer 2 (BD Biosciences) for 10 minutes at room temperature for permeabilization. Cells were then washed with wash buffer and stained with anti-IL-2 APC, anti-IFN-γ (PE CY7), and anti-CD3 (Amcyan), anti-CD4 (PE, D2), or anti-CD8 (Percp CY5.5) (BD Pharmingen). Forty thousand (40,000) lymphocytes per sample were obtained using LSR II (BD Biosciences) and analyzed using FLOWJO software (TreeStar). The percentage of cytokines producing CD4 or CD8 T cells without antigen stimulation was <0.05% (negative control group). Results are expressed as the percentage of CD4+ (or CD8+) T cells expressing IFN-γ or IL2.

[0487] 2.5. Statistical Analysis

[0488] Statistical analysis and comparison were performed using paired t-tests.

[0489] 3. Results

[0490] The results provided by this SEB antigen-induced T-cell proliferation assay show that, in patients not receiving HIV ART and age-consistent healthy individuals, mAb dB4C7 (1 µg / mL) and anti-HIV RC polyclonal antibody (25 µg / mL) at saturation reduced CD4+ T-cell proliferation, but not CD8+ T-cell proliferation (data not shown).

[0491] MAb dB4 (1 µg / mL) and purified anti-HIV RC polyclonal antibody (25 µg / mL) at their individual saturated PBMC surface CD4 binding concentrations, in HIV-negative ( FIG. 18A ) and HIV positive ( FIG. 18B In both individuals, IL-2 production in proliferating CD4+ T cells induced by the superantigen SEB was suppressed. This suppression was not observed in CD8+ T cells from the same HIV-positive and HIV-negative individuals. FIG. 18C ).

[0492] MAb dB4 (1 μg / mL) and purified anti-HIV RC antibodies (25 μg / mL) inhibited the production of IFN-gamma by proliferating CD4+ T cells triggered by the superantigen SEB at their individual saturating concentrations in HIV-negative FIG. 18D ) and HIV-positive FIG. 18E ) individuals. No such inhibition was found in CD8+ T cells from the same HIV-negative FIG. 18F ) and positive FIG. 18G ) individuals.

[0493] 4. Conclusion

[0494] The antibodies mAb dB4C7 (UB-421) and anti-HIV RC polyclonal antibodies, both directed against the CDR2 region of CD4 domain 1, were found to inhibit T cell proliferation and cytokine (IL2 and IFN-gamma) production by CD4-positive T cells triggered by the superantigen SEB, but not by CD8-positive T cells. The inhibition of CD4+ T cell proliferation and associated cytokine (IL2 and IFN-gamma) production by dB4C7 and anti-HIV RC polyclonal antibodies suggests that these antibodies can exert similar inhibitory effects on other CD4-positive cell-associated cytokine production, with the potential to disrupt the pathogenic cycle of pyroptosis by HIV.

[0495] As observed in the previous examples, the inhibitory effects on CD4-positive T cell proliferation and associated cytokine (IL2 and IFN-gamma) production were highly significant, where the antibodies directed against the CDR2 region described herein can exert simultaneous opposite effects on CD4 cells, including: (1) silencing the reactivation of HIV-infected CD4-positive T cells to trigger the release of HIV from their latent state (Example 9); (2) competitively inhibiting and preventing the new release of HIV from entering uninfected CD4-positive T cells by the reactivation of silenced CD4+ T cells (Examples 4 and 6); and (3) inhibiting T cell proliferation and cytokine production by CD4-positive T cells upon (super)antigen stimulation (this Example).

[0496] The unique biological profile and immunological responses elicited by mAb dB4 and anti-HIV RC polyclonal antibodies directed against specific locations of HIV binding, i.e., the CDR2 region of CD4 domain 1, can provide the necessary properties for a functional cure of HIV infection, i.e., the ability to (1) prevent HIV infection by entry inhibition; (2) manufacture virus upon silencing T cell reactivation; and (3) directly alter cytokine production.

[0497] Example 11. Inhibition of HIV-1 replication by mAb dB4C7 (UB-421) and anti-HIV RC polyclonal antibodies in primary isolates of clade B HIV-1DH12 Pre-exposure and post-exposure prophylaxis and treatment of HIV infection in challenged chimpanzees using mAb B4

[0498] B4-related high affinity antibodies directed against the CDR2 region of CD4 domain 1 have been shown to have a number of unique in vitro properties, it is important to test the efficacy of the B4 antibody in preventing and / or treating HIV infection in an animal model that most closely resembles humans. Chimpanzees have been used as a model for human viral, bacterial, and parasitic infections for over 100 years. In a carefully designed challenge model in chimpanzees, a primary isolate of HIV-1 from clade B, HIV-1 DH12 A challenge experiment was performed to evaluate the potential of mAb B4 to provide passive immunity against HIV-1 infection in both pre-exposure and post-exposure modes. Specifically, the ability of mAb B4 to provide (1) sterilizing immunity to protect exposed subjects against infection and (2) therapy to exposed subjects when the antibody is administered several days after confirmed infection was evaluated.

[0499] 1. Pre-exposure prophylaxis of HIV infection in chimpanzees using mAb B4

[0500] The potential of mAb B4 to provide passive immunity against HIV-1 infection in both pre-challenge and post-challenge (exposure to HIV-1) modes was evaluated.

[0501] 1.1. Methods

[0502] 1.1.1 Animals used in the experiment

[0503] A total of 4 chimpanzees were used in this experiment. One chimpanzee was used as a pre-exposure therapy animal (X084), two chimpanzees were used as post-exposure therapy animals (X356 and X357), and one chimpanzee was used as a control (X259).

[0504] 1.1.2 Sensitivity of the animals to infection with HIV-1 DH12 virus stock

[0505] The sensitivity of the animals to infection with HIV-1 DH12 virus stock using in vitro infection assays with their PBMCs. All cultures were infectable within 3 days of exposure to the virus.

[0506] 1.1.3 MAb B4 antibody

[0507] MAb B4 was prepared for infusion as a highly purified antibody preparation (5 mg / mL concentration).

[0508] 1.1.4 Pre-challenge prophylaxis / therapy

[0509] HIV-1 DH12 Chimpanzee X084 was intravenously infused with 5 mg / kg of mAb B4 one hour prior to challenge.

[0510] 1.1.5 Post-challenge prophylaxis / treatment

[0511] HIV-1 DH12 Chimpanzees X356 and X357 were intravenously infused with 5 mg / kg of mAb B4 one hour post-challenge.

[0512] 1.1.6 Control animals

[0513] HIV-1 DH12 Chimpanzee X259 was challenged without infusion of mAb B4 antibody.

[0514] 1.1.7 Challenge with HIV-1 DH12

[0515] HIV-1 DH12 Four chimpanzees were intravenously challenged with 100 TCID 50 of virus taken from a previously prepared stock and titered in chimpanzee PBMC at the Southwest Foundation for Biomedical Research.

[0516] 1.1.8 Detection of HIV-1 DH12 virus

[0517] Infection establishment in chimpanzees was monitored by detection of plasma viremia, cell-associated viral load and immune response to HIV by DNA PCR amplification of gag sequence, co-cultivation, p24 capture ELISA and immunoblotting. Serum viral load indicators of HIV infection were determined using HIV-1 RNA copies / mL from all blood samples collected during the 50-week study period in all chimpanzees. HIV-1 DH12 virus was detected in chimpanzee PBMC using virus isolation and DNA PCR assays (DNA PCR assays were used to detect proviral DNA corresponding to gag). Virus production was assessed using a p24 antigen capture ELISA (Coulter). Serial dilutions of 1 x 10 6 to 1 x 10 2 cells of chimpanzee PBMC and lymph node cells were co-cultivated with 2 x 10 6 cells of PHA-stimulated parental cells from day 3 in IL-2 medium. The well with the highest dilution that resulted in p24 production was selected as the endpoint indicator.

[0518] 1.1.9 Animal housing ​

[0519] Chimpanzees were housed at the Southwest Foundation for Biomedical Research with approval of the Institutional Animal Care and Use Committee and in accordance with the National Research Council's Guide.

[0520] 1.2. Results

[0521] Chimpanzees receiving mAb B4 against HIV-1 DH12 were protected from infection. In animals infused with mAb B4 prior to challenge with HIV-1 (X084) FIG. 19A ), or in two animals infused with mAb B4 one hour after challenge with HIV-1 (X356 and X357) FIG. 19B ), no markers of infection were detected during the 32 weeks following challenge.

[0522] In contrast, virus was readily isolated from PBMC FIG. 19C ) of control animals (X259) beginning at week 1 after challenge and from plasma at week 2. Virus was also isolated from lymph node cells of X259 at weeks 4 and 20. Infected cells were detected in PBMC and lymph node compartments in dilutions containing cell numbers ranging from 1 x 10 4 to 1 x 10 6 Serum conversion occurred in animal X259 at week 4.

[0523] The free, unbound mAb B4 present in the circulation of chimpanzees X084, X356 and X357 treated with antibody was found to decline rapidly. There was no evidence of CD4+ depletion in the CD4+ and CD8+ subsets from treated chimpanzees monitored during the 20 weeks following challenge. By week 32, chimpanzee PBMC had no inhibitory effect on the proliferative response to mitogens (PHA, pokeweed mitogen and Concanavalin A).

[0524] These results indicate that mAb B4 can provide a prophylactic or curative treatment for HIV infection. The results were confirmed by extensive follow-up of serum viremia and other parameters for up to one year from the day of challenge.

[0525] 1.3. Conclusion

[0526] In the chimpanzee experiments, mAb B4 administration prior to or within a short time interval after exposure abrogated HIV infection by the pathogenic primary isolate. Transferred immunity was eliminated, and there was no evidence of transient, reduced, or delayed viremia. The complete protection was evident despite the rapid clearance of mAb B4 antibody from the plasma, which was trapped on CD4+ cells in the peripheral blood and lymphoid tissues by mAb B4.

[0527] Well-conducted experiments further indicate that a therapeutic regimen using 5 mg / kg of mAb B4 or related antibodies can be used to achieve a curative cure in (i) HIV-infected patients within hours, or (ii) infants born to seropositive mothers at birth.

[0528] 1.4. Use of mAb B4 for post-exposure prophylaxis

[0529] The U.S. Public Health Service guidelines for health care workers accidentally exposed to HIV recommend post-exposure prophylaxis using anti-retroviral drugs. However, the U.S. Public Health Service guidelines are reserved about the toxicity of the currently available drugs for post-exposure prophylaxis.

[0530] The results obtained in this experiment demonstrate the potential of using mAb B4 as a post-exposure prophylactic treatment instead of or in combination with the current post-exposure treatment methods. The low toxicity and efficacy of mAb B4 demonstrate the potential of this antibody to be more broadly effective than anti-retroviral drugs.

[0531] These results further indicate that mAb B4 can be used for the prevention of HIV vertical transmission from mother to child. Mother-to-child transmission (MTCT) of HIV, also known as perinatal or vertical transmission, occurs during pregnancy, delivery, and / or during breastfeeding or mother's milk feeding, and transmits HIV from an HIV+ woman to her infant. The probability of MTCT for an HIV+ woman who is not receiving treatment for the virus during pregnancy, delivery, and birth is about 25%. In an untreated HIV+ woman who breastfeeds her infant, there is an additional 12% chance of MTCT. Worldwide, in 2001, 1.8 million women were infected with HIV, and approximately 8 million children also became HIV infected, most of them via MTCT. The majority of people newly diagnosed with HIV worldwide are between 15-24 years of age. A very important component of MTCT prevention is that HIV prevention must be directed at young people, especially girls and young women, before they become sexually active, and treatment of those who are infected. Additional information on MTCT can be found at http: / / caps.ucsf.edu / archives / factsheets / mother-to-child-transmission-mtct#sthash.DTRyms46.dpuf. The results obtained with mAb B4 indicate that MTCT can be prevented during the delivery and birth stages by providing a single administration of 5 mg / kg or more of mAb dB4 to the newborn of an HIV+ woman.

[0532] 2. Treatment of HIV infection in chimpanzees with mAb B4

[0533] 2.1. Methods

[0534] 2.1.1 Animals used in the experiments

[0535] Chimpanzees X084, X356, and X357 were used in a previous prevention experiment and received a single administration of 5 mg / kg of mAb B4 to protect them from infection with HIV-1 DH12 which were used again in this challenge experiment in a therapeutic mode.

[0536] 2.1.2 Sensitivity of the animals to infection with HIV-1 DH12 virus stock

[0537] PBMCs were prepared from the animals prior to antibody treatment and HIV challenge to determine their sensitivity to infection with HIV-1 DH12 All in vitro cultures were infected with the virus within 3 days of the vaccination.

[0538] 2.1.3 MAb B4 Antibody

[0539] MAb B4 was prepared for infusion as a highly purified antibody preparation (5 mg / mL concentration).

[0540] 2.1.4 Treatment with HIV-1 DH12 Challenge

[0541] HIV-1 DH12 at 100 TCID 50 (derived from a previously prepared viral stock and titered in chimpanzee PBMCs at the Southwest Foundation for Biomedical Research) was intravenously challenged in all animals (X084, X356 and X357).

[0542] 2.1.5 Post-Challenge Treatment

[0543] Chimpanzee X084 received no post-challenge treatment. Chimpanzee X357 was infused with 5 mg / kg of MAb B4 on day 14 post-challenge, at which time viremia in the challenged animal treated for HIV infection was at its highest. Chimpanzee X356 was infused with 5 mg / kg of MAb B4 on days 14, 18 and 22 post-challenge.

[0544] 2.2. Summary of Results

[0545] A passive immunotherapy trial was performed using infusions of 5 mg / kg of murine MAb B4 to determine the efficacy of MAb B4 in chimpanzees with acute HIV infection. HIV-1 DH12 Three chimpanzees (X084, X356 and X357) were intravenously (i.v.) vaccinated and infection was confirmed based on viral detection (not shown) and quantitative RT-PCR.

[0546] RNA from HIV-1 DH12 was detected in all chimpanzees (X084, X356 and X357) by RT-PCR 7 days post-exposure (above baseline). Cell-associated virus was detected in PBMCs and lymph node cells from all three animals by 14 days using viral isolation and DNA-PCR for HIV-1 DH12 integration detection.

[0547] At day 14 post-infection, two of the infected chimpanzees (X356 and X357) were infused with mAb B4 (5 mg / kg, i.v.). At days 18 and 22 post-infection, one animal (X356) received two additional doses of antibody (5 mg / kg, i.v.). Viral load was monitored by RT-PCR assay at weekly intervals for the first 12 weeks and then monthly until 40 weeks post-infection. At day 14 post-infection, when chimpanzees X356 and X357 were infused with mAb B4, viral RNA was already in a rapid rise phase. In contrast to chimpanzee X084 (which did not receive treatment) ( FIG. 20A , open circles), the two chimpanzees that received antibody then experienced a rapid and significant drop in their viral load (1-2 logs) at day 20 post-infection, as well as a significant reduction in the duration of the initial viremic phase (from 4 weeks to 1 week) FIG. 20A , closed circles).

[0548] FIG. 20A and FIG. 20B The duration of plasma viremia in chimpanzee X356, which received three infusions of mAb B4, was compared to chimpanzee X084 (from this experiment, which received a single pre-exposure dose of mAb B4) and chimpanzee X259 (from a previous experiment, which did not receive mAb B4). Following the viral challenge, serum HIV-1 RNA copies were measured without any intervention using HIV-1 DH12 Chimpanzees X084 ( FIG. 20A , open circles) and X259 ( FIG. 20B , open circles) were challenged with HIV-1 and showed viremia as early as day 3. The duration of serum HIV-1 viremia was approximately 42 days, which is characteristic of HIV-1 DH12 Chimpanzee X356 ( FIG. 20A and 20B , closed circles) received three doses of mAb B4 and HIV-1 viremia dropped sharply, reaching below the limit of detection at or around day 21. Comparing the viral load data between X356 and X084 and X259 shows that the duration of viremia, which is characteristic of HIV-1 DH12 infection, was reduced by 21 days.

[0549] As mentioned above, chimpanzee X084, which was from this experiment and served as an untreated control, received a single pre-exposure dose of mAb B4 in a previous experiment; chimpanzee X259, which was from a previous experiment and served as an untreated control, did not receive any mAb B4. Interestingly, comparing X084 ( FIG. 20A , open circles) and X259 ( FIG. 20BThe data provided by the open circles show that X084 had a lower overall viral load during the experiment than X259. Thus, while the viral load of the untreated animal (X084) in this experiment was significantly higher than that of the mAb B4 treated animal (X356), prior exposure to mAb B4 in X084 appears to have significantly reduced the overall viral load, particularly during the acute phase of HIV infection (compare X084 with X259). Overall, these results indicate that any administration of mAb B4 prior to HIV exposure can reduce the extent of viral load during the acute phase of infection, which can further reduce the transmission of the virus from individual to individual after infection.

[0550] In addition, cell surface staining was performed and found that mAb B4 binding to CD4+ cells persisted for at least seven days in animals (X357) administered a single dose of mAb B4 on day 14. And mAb B4 binding to CD4+ cells persisted for at least 14 days in animals (X356) administered multiple doses of the antibody. In contrast, free mAb B4 was detected in the blood circulation only in animals three days after infusion, as measured by an ELISA assay for HIV-1 specific antibodies. DH12 The neutralizing activity of the isolates was measured as described above. As in the prevention experiment in chimpanzees, this is consistent with mAb B4 having been removed from the blood circulation by binding to CD4+ cells.

[0551] FACS analysis using in vitro immunostaining for the CD4 / B4 epitope detected CD4+ cells in all samples without significant depletion during the 40 day period. Immunostaining for mAb B4 was performed on PBMC during the first 21 days. The mitogen-induced proliferative response of PBMC samples (using concanavalin, PWM or PHA) was different before and after antibody treatment and did not appear to be affected by mAb B4 infusion. These observations indicate that mAb B4 is able to destroy virus infection and reduce viral load and viremic phase without undue immunotoxicity.

[0552] Example 12. UB-421 formulation, preclinical pharmacology / toxicity testing in baboons and cross-reactivity in human tissues

[0553] SUMMARY

[0554] The data obtained in the cell culture and chimpanzee experiments discussed in the foregoing examples demonstrate sufficient scientific merit to justify further development of a pharmaceutical preparation containing mAb dB4 for use in humans. A pharmaceutical preparation containing mAb dB4 C7 (UB-421) was prepared and subjected to general safety testing on a large scale for clinical use.

[0555] In addition, a set of preclinical safety tests were performed to provide pharmacodynamic, pharmacokinetic, toxicity, and safety information for the candidate drug UB-421.

[0556] Single and multiple dose and dose-dependent (low and high dose) tests were performed in Papio species to assist in the design of the Phase I clinical trial to assess the dose-dependent safety, tolerability, and immunotoxicity of UB-421 in asymptomatic HIV-1 infected human subjects. Papio species were used as an animal model in the preclinical pharmacology / toxicity tests because the CD4+ T cells of Papio species have similar binding affinity to mAb dB4C7 as humans, as shown in FIG. 21 (i.e., EC 50 = 0.33 pg / mL and 0.29 pg / mL, respectively). Dr. Krishna Murthy (Southwest Foundation for Biomedical Research, Department of Virology and Immunology, San Antonio, TX) was contracted to initiate and perform the non-human primate pharmacology and toxicity tests in Papio species.

[0557] In addition, cross-reactivity tests were performed to assess whether UB-421 has any unintended reactivity and possible sites of cytotoxicity to human tissues other than the intended target.

[0558] As discussed in further detail below, the data obtained from these preclinical tests showed sufficient scientific merit to justify further development of UB-421 as an investigational new drug in human clinical trials.

[0559] 1. Pharmaceutical dosage form

[0560] Pharmaceutical dosage forms containing mAb dB4C7 were prepared for use in humans. In general, pharmaceutical dosage forms containing mAb dB4C7 can be formulated in an appropriate buffer including, but not limited to, citrate, phosphate, Tris, BIS-Tris, etc., at a pH value between 6.0 and 7.0, and can contain excipients (e.g., a sugar (50 mM to 500 mM sucrose, trehalose, mannitol, or a mixture thereof), a surfactant (e.g., 0.025% - 0.5% Tween 20 or Tween 80), and / or other agents.

[0561] UB-421 is a pharmaceutical composition designated to contain 10 mg / mL mAb dB4C7, 20 mM glycine, and 0.05% (v / v) Tween (polysorbate 20) in phosphate buffered saline (PBS) (pH 6.5).

[0562] High concentration formulations of MAb dB4 can also be prepared for use involving subcutaneous injection, including 10 mM histidine.

[0563] Following manufacture, general safety and toxicity tests are performed to ensure that the manufactured drug product is safe for administration to human and animal subjects.

[0564] 2. General Safety Tests

[0565] 2.1. UB-421 Production Lots and Safety Criteria

[0566] UB-421 drug product was manufactured for clinical testing in two large scale production runs (P / N Z807, Lots 225711 and 225758) and evaluated for general safety.

[0567] A large scale production run of UB-421 is considered safe and acceptable for clinical use if the following conditions are met during the 7 day test period: (1) all animals survive the entire test period; (2) no overt signs of toxicity are observed; and (3) no animal has a significant body weight loss during the period from the time of administration of the pharmaceutical composition until the end of the test period.

[0568] 2.2. Test Animals

[0569] Mice: Albino, BALB / cByJNarl strain (Mus musculus), specific pathogen free, male (Taiwan). (Experiment No. BIO-003.90).

[0570] Guinea pigs: Albino, Hartley strain (Cavia porcellus), specific pathogen free, male (National Taiwan University Hospital, Animal Center, Taiwan). (Experiment No. BIO-003.91).

[0571] 2.3. Methods

[0572] Each lot of UB-421 was injected intraperitoneally into two mice and two guinea pigs. Control mice and guinea pigs were injected intraperitoneally with isotonic sodium chloride solution ("S.T.", Lot COC0206). Each mouse received a total volume of 0.5 mL, and each guinea pig received a total volume of 5.0 mL. Animal body weights were recorded prior to dosing (Day 0) and at the end of the test (Day 7). Each animal was observed daily for general health and clinical signs of toxicity.

[0573] 2.4. Analysis and Conclusion

[0574] The results from the general safety test resulted in the following satisfactory results against the analysis criteria:

[0575] (1) All animals used for the general safety test survived the test period.

[0576] (2) No overt signs of toxicity were observed in UB-421 treated animals.

[0577] (3) No body weight loss in animals during the test period.

[0578] In view of the above, these batches are considered free of unexpected, unacceptable foreign impurities.

[0579] These findings confirm that the large scale manufacturing batches of UB-421 (batch numbers 225711 and 225758) are free of unexpected or unacceptable foreign impurities. Accordingly, both batches are considered safe and acceptable for use in human clinical trials.

[0580] 3. Test A: Single administration toxicity test of UB-421 in baboons

[0581] 3.1. Methods

[0582] Test A evaluated the pharmacodynamics, pharmacokinetics and safety of a single administration of UB-421 at a low dose (5 mg / kg body weight) or a high dose (25 mg / kg body weight) over a period of 42 days, as shown in Table 14A. In this test, UB-421 was administered as an intravenous infusion over a 30 minute period at 5 mg / kg or 25 mg / kg body weight. Blood samples for pharmacokinetic (PK) analysis were collected at 0, 0.5, 1, 2, 4, 8, 12, 24 hours, and on days 2, 3, 5, 7, 10, 14, 21, 28, 35, 42.

[0583] Blood samples were analyzed using flow cytometry analysis to monitor the presence of CD4+ T lymphocytes using the following markers, including: CD4 domain 1 (anti-CD4+D1) and CD4 domain 2 (anti-CD4+D2). Alexa-dB4 was used as a marker and as a tracer for competitive CD4 binding (competitive CD4 binding is with monoclonal antibody UB421, and both monoclonal antibody UB421 and Alexa-dB4 are directed to CD4 domains 1 and 2). Alexa-goat anti-huIgG was used as a tracer for the monoclonal antibodies directed to CD4 domains 1 and 2. In addition, antibodies directed to CD3 and CD14 were used to monitor the total T cell (CD3) and monocyte (CD14) numbers in the PBMC preparation.

[0584] 3.2. Brief summary of results

[0585] No adverse findings were observed in the pharmacodynamics, pharmacokinetics and safety tests that would preclude UB-421 from being taken to Phase I clinical trials in humans. For all parameters evaluated, significant findings that were significantly higher or lower than the mean ("out of range") were observed in both control and experimental animals.

[0586] The initial pharmacokinetic (PK) properties of UB-421 were evaluated on blood samples provided from baboons treated with a single administration of UB-421 at low (5 mg / kg) and high (25 mg / kg) doses.

[0587] The results of this test showed that treatment with UB-421 resulted in a significant decrease in the detection of CD3+CD4+ T lymphocytes and CD14+CD4+ monocytes from either peripheral blood mononuclear cells (PBMC) or cell suspensions from lymph node biopsy samples. It was hypothesized that the decrease in these cells was due to "covering" of the CD4+ cells with UB-421 and not because of depletion of CD4+ cells from the animal. This hypothesis was confirmed by finding that the percentage of CD4+ T lymphocytes detected did not change when using antibodies against anti-CD4+ D2 markers. The duration of "covering" of CD4+ cells with UB-421 after infusion was at least 3 days in animals receiving the low dose and at least 7 days in animals receiving the high dose. When UB-421 was no longer detected in the plasma of the animals, the "covering" of CD4+ cells decreased and the CD4+ cells (as detected with anti-CD4+ Dl) returned to the same percentage as detected with anti-CD4+ D2.

[0588] In summary, this study found that UB-421 is safe and that UB-421 can completely cover CD4 positive cells for at least 3 days when administered at a low dose and for at least 7 days when administered at a high dose. Based on the results discussed in the previous examples, when CD4+ cells are completely covered, it is expected that HIV entry into the cell will be completely excluded, which will result in a decrease in viral load.

[0589] 4. Test B: Multiple administration toxicity test in baboons

[0590] 4.1. Methods

[0591] Trial B evaluated the pharmacodynamics, pharmacokinetics, and toxicity of repeated administration of UB-421 at a low dose (5 mg / kg body weight) or a high dose (25 mg / kg body weight) for a period of 56 days (8 weeks) as shown in Table 14B. In this trial, one treatment period consisted of 8 weekly doses of UB-421 (one dose per week). At the end of the 8-week treatment period, half of the treated animals were necropsied and evaluated (Table 14B, Part 1). The remaining animals were then maintained for a 12-week recovery period during which they were observed and blood and lymph node samples were collected (Table 14B, Part 2). Blood samples (and lymph node biopsies) were collected from the animals for analysis at 0, 1, 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 91, 105, and 133 days during the treatment period and recovery period. This trial system used adult male (M) and female (F) baboons (n = 20; age: 7 to 18 years).

[0592] 4.2. Results

[0593] Pharmacokinetic, pharmacodynamic, toxicity, and safety information from a battery of large-scale preclinical trials of multiple doses of UB-421 at a low dose (5 mg / kg) and a high dose (25 mg / kg).

[0594] Overall, the data provided by Trial B, Part 1 and Part 2 indicate that the UB-421 candidate drug has scientific value for further development as a test article. No negative findings were discovered in the pharmacodynamic, pharmacokinetic, and safety studies that would preclude UB-421 from proceeding to Phase I clinical trials in humans. For all parameters evaluated, significant findings above or below the mean ("out of range") were observed for both control and experimental animals.

[0595] 4.2.1 Blood and biopsy analyses

[0596] A battery of large-scale tests was performed on samples obtained from animals treated with multiple doses of UB-421 at a low dose and a high dose. The results of these tests are summarized in Tables 15 and 17 and discussed further below.

[0597] Using the UBI ELISA test (Example 7) and the MT-2 test (Example 2), UB-421 was detected in the plasma of animals receiving the low dose (5 mg / kg) for at least 3 days and in the plasma of animals receiving the high dose (25 mg / kg) for at least 7 days. In some animals receiving the high dose, excess UB-421 was detected in the plasma for at least 14 days after the last infusion.

[0598] No baboon antibodies to UB-421 were found in any of the 16 animals that received UB-421, confirming that this candidate drug was not immunogenic in the treated animals.

[0599] All baboons were immunized with hepatitis B virus (HBV) vaccine (Merck) on days 0 and 28. Detectable levels of baboon anti-HBsAg antibodies were observed on day 56 in 4 of 4 control (1B) animals, 6 of 8 baboons receiving low dose (2B) UB-421, and 3 of 8 baboons receiving high dose (3B) UB-421. These results suggest that "coverage" of CD4+ cells by UB-421 can cause some animals to be less responsive to antigen prophylaxis. However, the effect of this reduced responsiveness was found to be reversible, as all of these animals were able to develop anti-HBsAg antibodies after discontinuation of UB-421 treatment and additional immunization with HBV vaccine.

[0600] 4.2.2 Results of Observations and Necropsy

[0601] The gross and microscopic analyses of the treated baboons in this study are summarized in Tables 16 and 17.

[0602] In general, the tissues evaluated in this study from all baboons (Parts 1 and 2) did not show any unique or consistent pathological changes that could be attributed to administration of UB-421 at either the low or high dose level.

[0603] Ophthalmic observations were performed on all Group 3B (high dose) animals (n=8) at the time of admission prior to infusion and again at the end of the treatment period, one week after the last infusion. The ophthalmic reports from these animals indicate that both eyes remained within normal limits for all tests performed at the end of the treatment period.

[0604] Electrocardiographic observations were obtained from all animals (n=20) before, during, and after the completion of each dose administration. The electrocardiographic reports from all test animals concluded that the changes found were within the range of normal day-to-day variation.

[0605] 4.2.3 Results of IND-Enabling Toxicology Studies

[0606] There were no significant differences in immunotoxicity results between the UB-421 treatment groups (2B and 3B) and the control group (IB) in addition to the decreased immune response to HBV vaccination. Clinical examination results, ophthalmology reports, electrocardiogram recordings, and histopathology results support the conclusion that UB-421 is safe and well-tolerated in adult baboons after receiving 8-week infusions at dose levels up to 25 mg / kg, and that the therapeutic method effectively covers the target cells without causing cell depletion.

[0607] 5. Cross-reactivity in human tissues

[0608] As discussed in the previous example, the mAb dB4 antibody, including mAb dB4C7 (the primary component of the UB-421 candidate drug), demonstrated high binding affinity for CD4, particularly membrane-bound CD4 located on T cells. The following preclinical study utilized an array of 30 human organ tissues to assess whether mAb dB4C7 could bind to other cell types. The objective of this study was to assess whether mAb dB4C7 had any unexpected reactivity and potential sites of cytotoxicity to cells other than the intended target human tissues.

[0609] 5.1. Methods

[0610] An immunohistochemistry study was performed using an FDA-standard frozen tissue array containing 90 tissue cores from 30 organs, each tissue taken from 3 normal human individual donors (BioMax Tissue Microarray (TMA) sections, Rockville, MD). Additional frozen sections from individual human samples were also included in the human tissue survey, as described in Table 18. The human tissue survey was screened for immunoreactivity with biotinylated mAb dB4C7 and control reagent (biotinylated goat anti-rabbit IgG) to assess specificity and unwanted autoreactivity.

[0611] 5.2. Results

[0612] The staining pattern observed on adult normal tissue sections was reviewed by a board-certified clinical pathologist at PhenoPath Laboratories (Seattle, WA) and scored for reactivity. Strong, surface cell membrane staining was observed in the thymus and T cell dependent areas located in the tonsil (and lymph node), with weak positive reactivity noted in the spleen. Occasional mononuclear cells and lymphocytes or macrophages stained positively in a predictable pattern in multiple tissues. Localized weak Kupffer cell staining was also noted in the liver. All other human tissues tested were negative, except for weak staining of endogenous biotin in certain tissues. Pre-incubation of the tonsil section with the unlabelled mAb dB4C7 antibody blocked the specific staining pattern. No unexpected cross-reactivity was observed.

[0613] These results demonstrate that mAb dB4C7 (the major component of UB-421) does not have any unexpected cross-reactivity that could lead to cytotoxicity in human tissues other than the intended target.

[0614] Example 13. Phase I, open-label, single-dose, dose-dependent trial to assess the safety and pharmacokinetics of UB-421 in asymptomatic HIV-1 infected adults

[0615] 1. Objectives

[0616] The primary objective was to assess the safety and tolerability of a single intravenous infusion of UB-421 at escalating doses in subjects with asymptomatic human immunodeficiency virus-1 (HIV-1) infection. The secondary objective was to determine the pharmacokinetics of a single intravenous infusion of UB-421 at escalating doses in subjects with asymptomatic HIV-1 infection. (ClinicalTrials.gov Identifier: NCT01140126).

[0617] 2. Methods

[0618] Open-label, single-dose, dose-dependent (dose-escalation), two-center, non-comparative.

[0619] 3. Number of subjects

[0620] A total of 20 subjects (5 subjects per dose cohort) were enrolled.

[0621] 4. Diagnosis and main inclusion criteria

[0622] To be eligible for participation in this trial, subjects must have met all of the following criteria.

[0623] (1) HIV-1 seropositive;

[0624] (2) Age 20 years or older;

[0625] (3) For protocol version Mar-09-2010: asymptomatic, as determined by the Investigator at Screening Visit (Visit 1, VI) based on medical history, physical examination, electrocardiogram (ECG), and coagulation tests and clinical chemistry and hematology test results, which must remain within the normal range ± 10% throughout the study;

[0626] (4) For protocol version Jul-01-2010: asymptomatic, defined as no acute or symptomatic viral hepatitis and no history of AIDS-defining illness within 24 weeks prior to Screening Visit (Visit 1, VI), as determined by the Investigator based on medical history, physical examination, ECG, and laboratory values;

[0627] (5) For protocol version Mar-09-2010: CD4+ T cell count > 350 cells / mm 3 and HIV-1 viral load > 5,000 copies / mL obtained at Screening Visit (Visit 1, VI);

[0628] (6) For protocol version Jul-01-2010: CD4+ T cell count > 350 cells / mm 3 and HIV-1 viral load > 5,000 copies / mL obtained within 4 weeks prior to Screening Visit or at Screening Visit (Visit 1, VI);

[0629] (7) Treatment-naive, i.e., subjects who have not received antiretroviral therapy prior to and at the time of the study;

[0630] (8) Non-breast-feeding women;

[0631] (9) Subjects must have a negative serum pregnancy test result at the Screening Visit for women of childbearing potential;

[0632] (10) Subjects must agree to use a barrier method of contraception (female or male condom) during the study period; and

[0633] (11) Subjects should sign an informed consent form prior to receiving any study procedures.

[0634] 5. Test Drug and Intervention Methods

[0635] UB-421 (dB4C7 mAb) is supplied at a concentration of 10 mg / mL (100 mg in a 10 mL vial).

[0636] Subjects were assigned to 4 dose groups, each containing 5 subjects, based on the dose of UB-421 they received. Each enrolled subject received a single intravenous infusion of UB-421 at one of the following dose levels on Day 0 (Visit 2, V2): 1 mg / kg body weight (Dose 1), 5 mg / kg body weight (Dose 2), 10 mg / kg body weight (Dose 3), or 25 mg / kg body weight (Dose 4). The appropriate volume of UB-421 was calculated based on the specific dose and subject body weight. The volume of each individual dose was adjusted using sterile normal saline, whereby each individual subject within a dose group was infused with an equivalent volume of drug infusion. The dose of UB-421 was then delivered using an infusion pump.

[0637] 6. Treatment: 1 course

[0638] From the time frame of screening, treatment, and follow-up: 62 to 90 days. From the time of infusion to the end of the trial: 60 days.

[0639] 7. Evaluation Criteria:

[0640] 7.1. Primary Safety Endpoint:

[0641] (1) Physical Examination (PE)

[0642] (2) Vital Signs

[0643] (3) Clinical Chemistry and Hematology Tests

[0644] (4) Incidence of adverse events (AEs) / serious adverse events (SAEs)

[0645] (5) Electrocardiogram (ECG)

[0646] 7.2. Secondary Safety Endpoints:

[0647] (1) Serum concentration of anti-UB-421 antibodies exhibited on Day 0 (pre-infusion), Day 14, Day 28, and Day 60 post-dosing (immunogenicity of UB-421).

[0648] (2) Initial CD4+ T cell count was assessed at Screening Visit (V1) or was based on pre-screening data provided within 4 weeks prior to the Screening Visit.

[0649] (3) Follow-up CD4+ T cell count was assessed on Day 60 post-dosing.

[0650] 8. Efficacy Endpoint

[0651] The efficacy endpoint was assessed as the change from baseline in HIV-1 viral load. Baseline HIV-1 viral load was defined as the viral load assessed at Screening Visit V1.

[0652] 9. Pharmacokinetics (PK)

[0653] The following pharmacokinetic (PK) parameters were assessed for UB-421:

[0654] (1) Cmax: the highest serum drug concentration observed after dosing. max

[0655] (2) Tmax: the time to reach Cmax. max max

[0656] (3) λz: the elimination (terminal) rate constant. z

[0657] (4) t½: the elimination (terminal) half-life. 1 / 2

[0658] (5) AUC: the area under the serum drug concentration-time curve from time zero to the assessment time of the final sample. (0→last)

[0659] (6) AUC: the area under the serum drug concentration-time curve from time zero to infinity. (0→∞)

[0660] (7) CL: the serum clearance rate of drug from the body. β

[0661] (8) Vd: the volume of distribution in the elimination phase. ss

[0662] (9) Vss: the volume of distribution at steady-state. (0→∞)

[0663] (10) MRT: the mean residence time (MRT) extrapolated to infinity. (0→t)

[0664] 10. Statistical Methods

[0665] 10.1. Primary Safety Endpoints

[0666] (1) Physical Examination: physical abnormalities were summarized using narrative statistics by center, day, dose group, or overall. Tables of changes from baseline to the final visit were also presented.

[0667] (2) Vital Signs: vital signs were summarized using narrative statistics by each visit. Changes from baseline values were also presented by center, day, dose group, or overall.

[0668] ​​​​​​​​​​​(3) Clinical Chemistry and Hematology Tests: Results of clinical chemistry and hematology tests are summarized by descriptive statistics at applicable visits. Test results are categorized into four categories according to the Investigator's professional judgment and normal range criteria at each study site: normal, abnormal but not clinically significant (NCS), abnormal but clinically significant (CS), and abnormal and typically clinically significant (CST). Changes in laboratory test results from baseline to the final visit are also presented in a transition table.

[0669] (4) Incidence of Adverse Events (AEs): Pre-treatment adverse events, treatment-emergent adverse events (TEAEs), and drug-related adverse events are summarized by frequency distribution. Each reported adverse event is associated with an International

[0670] (5) Electrocardiograms (ECG): Electrocardiogram recording data provided at applicable visits are summarized by descriptive statistics.

[0671] 10.2. Secondary Safety Endpoints

[0672] (1) Serum Concentration of Anti-UB-421 Antibody: Antibody concentrations are summarized by applicable visits.

[0673] (2) CD4+ T Cell Counts: Cell counts and cell percentages are summarized by distribution statistics at applicable visits. Changes from baseline to the final visit in CD4+ T cell counts and percentages are also summarized.

[0674] 10.3. Efficacy Endpoints

[0675] (1) HIV-1 Viral Load: HIV-1 viral load is summarized by descriptive statistics at each visit. Changes from baseline values are also presented by center, day, treatment dose group, and overall = (value at post-treatment visit - value at baseline visit).

[0676] 10.4. Pharmacokinetic Assessments

[0677] Pharmacokinetic parameters are summarized by descriptive statistics at each visit.

[0678] According to the protocol and statistical analysis protocol (SAP), the safety endpoint was analyzed in the intent-to-treat (ITT) population; however, the efficacy analysis was conducted in both the ITT and efficacy populations. Regarding pharmacokinetic assessment, since samples were only available for PK data analysis from subjects at Taipei Veterans General Hospital (TVGH), these subjects were defined as the PK population. PK assessment was performed only on subjects at TVGH.

[0679] 11. Summary of Conclusions

[0680] 11.1. Efficacy, pharmacokinetics, and safety results

[0681] 11.1.1 Therapeutic Results

[0682] like FIG. 22A As shown, the efficacy of UB-421 was assessed by measuring changes in HIV-1 viral load at different time points throughout the trial. Efficacy analysis showed a significant net change in mean HIV-1 viral load after UB-421 infusion in subjects from the second (5 mg / kg), third (10 mg / kg), and fourth (25 mg / kg) dose groups, but not in the first (1 mg / kg) dose group.

[0683] The viral load decreased to 2.25 logs after a single administration. 10 (From the 10 mg / kg group) demonstrates the efficacy of the antibody drug UB-421 (mAb dB4C7). FIG. 22B The maximum average decrease in HIV-1 viral load for each dose group is summarized below, and... FIG. 22B show︰

[0684] Group 1 (1 mg / kg): 0.29 log on day 6 (V5). 10 1 copy / mL.

[0685] Group 2 (5 mg / kg): 0.97 log on day 6 (V5). 10 1 copy / mL.

[0686] Group 3 (10 mg / kg): 1.58 log on day 10 (V6). 10 1 copy / mL.

[0687] Group 4 (25 mg / kg): 1.63 log on day 14 (V7). 10 1 copy / mL.

[0688] The duration of HIV-1 suppression was dose-related, with subjects in the higher-dose group showing a longer duration of HIV-1 viral load reduction compared to those in the lower-dose group. FIG. 22A The longest duration of HIV-1 viral load suppression was observed in subjects who received the highest dose of UB-421 (dose group 4, 25 mg / kg), lasting approximately 28 days.

[0689] In summary, UB-421 antibody was found to have a strong antiviral effect in a dose-dependent manner at dose levels of 5, 10, and 25 mg / kg. That is, at comparable dosages, a higher percentage of patients treated with UB-421 achieved serum HIV-1 RNA levels >1 Log₂O₅ compared to those treated with TMB-355. 10 The reduction.

[0690] The previously reported efficacy data assessment of TMB-355 (formerly known as TNX-355; Kuritzkes, DR, et al., 2004, Figure 1) is based on a theoretical comparison using efficacy data from UB-421 in this trial, such as... FIG. 23A to FIG. 23C As shown. Based on this comparison, in 100% of the evaluated subjects, UB-421 achieved a 10-fold reduction in viral load when administered as a single dose of 10 mg / kg or 25 mg / kg; however, TMB-355 only achieved a 10-fold reduction in viral load in 83% of the subjects using the same dose.

[0691] 11.1.2 Pharmacokinetic Results

[0692] The pharmacokinetic (PK) parameters listed above were evaluated for each dose (1, 5, 10, and 25 mg / kg) of UB-421 used in this trial. Table 19 summarizes the multiple PK parameters (Cp) evaluated from 3 subjects in each dose group. max AUC (0→∞) T 1 / 2 The results (and MRT) showed a correlation between each PK parameter and the data value of the UB-421 dose. That is, increasing the UB-421 dose from 1 mg / kg to 25 mg / kg corresponds to an increase in the assessed PK parameter. Specifically, C max The concentration increased from 28.6 μg / mL to 462.5 μg / mL; AUC (0→∞) Increased from 201 μg-h / mL to 51367 μg-h / mL; T 1 / 2 The time increased from 14.4 hours to 85.4 hours; and the MRT (0→∞) It increased from 21.6 hours to 97.4 hours.

[0693] 11.1.3 Safety Results

[0694] The safety profile of UB-421 was assessed through physical examinations, vital signs, clinical chemistry and hematology tests, incidence of AEs / SAEs, and electrocardiograms. CD4+ T cell counts and anti-UB-421 antibody concentrations were also determined to provide further safety assessments.

[0695] The net incidence of TEAEs was 65.0% (13 of 20 subjects) across the total of 30 events, graded by severity. Three subjects reported six treatment-related adverse events with severity grading: grade 1 (mild) “pruritus” and “furunculosis” in subject B-001-001 (UB-421 at a dose of 1 mg / kg); grade 1 (mild) “increased lymphocyte count,” “decreased neutrophil count,” and “decreased platelet count” in subject A-015-009 (UB-421 at a dose of 10 mg / kg); and grade 2 (moderate) “measles-like rash” in subject B-015-008 (UB-421 at a dose of 25 mg / kg). The occurrence of the measles-like rash in subject B-015-008 was a Suspected Unexpected Serious Adverse Reaction (SUSAR); this subject was discharged from the hospital after 5 days of care. One subject reported an additional SAE that was not related to UB-421, an anal fistula and hemorrhoids in subject A-012-005 (UB-421 at a dose of 5 mg / kg); no treatment-related abnormalities were found in the results of vital signs or electrocardiograms. Only one dose was administered per treatment; therefore, no treatment discontinuations or dose changes occurred.

[0696] Anti-UB-421 antibodies were detected in three subjects (one from each of the 2nd, 3rd, and 4th dose groups) at Day 14 (V7) at levels slightly above the experimental limit of detection of 0.4 µg / mL. Anti-UB-421 antibodies were not detected at any subsequent visits through Day 60 (end of the trial). No related adverse events or other physiological abnormalities were associated with the presence of anti-UB-421 antibodies.

[0697] In addition, CD4+ T cell counts and percentages were relatively stable during the 60-day treatment period and the post-treatment follow-up period.

[0698] In summary, UB-421 was safe and well tolerated in HIV-1 -infected adults when administered as a single dose via intravenous (IV) infusion at a dose range of 1 to 25 mg / kg.

[0699] 12. Conclusion

[0700] This Phase I trial, with single intravenous infusions of UB-421 at dose levels ranging from 1 to 25 mg / kg, demonstrated that UB-421 is safe and well tolerated in HIV-1 infected adults. Most adverse events were mild or unrelated to the study drug. The only SUSAR of a measles-like rash occurred in the fourth dose cohort (25 mg / kg dose), but it is currently unclear whether this event is related to the study drug. Transient immune responses to UB-421 were detected in only three subjects at Day 14 (V7), with antibody levels only slightly above the assay limit of detection of 0.4 μg / mL, demonstrating that the appearance of this antibody is of minimal clinical significance. With respect to the pharmacokinetic profile of the study drug, the trends in the main parameters were related to the dose level. In addition, the degree and duration of HIV-1 viral load suppression were significantly positively correlated with UB-421 dose levels of 5, 10, and 25 mg / kg, but not as evident in the 1 mg / kg dose cohort.

[0701] In view of the safety and efficacy results obtained from this trial, further development of UB-421 at a dose level of at least 5 mg / kg is warranted for the treatment of asymptomatic HIV-1 infected adults.

[0702] Example 14. Phase Ila, open-label, multiple-dose, dose-dependent trial to investigate the safety and efficacy of UB-421 in asymptomatic HIV-1 infected adults

[0703] 1. Objectives of the trial

[0704] (1) To assess the safety and tolerability of multiple doses of UB-421 in a two-dose treatment regimen in HIV-1 infected subjects.

[0705] (2) To obtain evidence of antiviral activity of multiple doses of UB-421 in a two-dose treatment regimen in these subjects.

[0706] (3) To assess the antiviral activity and safety profile to determine the optimal UB-421 dosing and treatment regimen.

[0707] (ClinicalTrials.gov Identifier: NCT01668043).

[0708] 2. Trial design

[0709] This is an open-label trial utilizing repeat intravenous administration of UB-421. Subjects who are seropositive for HIV-1 and asymptomatic are screened for eligibility. Twenty-nine enrolled subjects receive multiple infusions of the study drug (UB-421) at one of two dose levels (10 mg / kg weekly (Dose Group 1) or 25 mg / kg every two weeks (Dose Group 2)) for an 8-week treatment period. Subjects are assigned to one of the two trial dose groups by site and rotation based on enrollment sequence. Subjects are followed for an additional eight weeks after the 8-week treatment period. The trial ends at Week 16.

[0710] 3. Inclusion Criteria

[0711] Subjects must meet the following criteria to be eligible for participation in the Phase Ila trial:

[0712] (1) asymptomatic, antiretroviral therapy (ART)-naive, HIV-1 seropositive

[0713] (2) CD4+ T cell count > 350 cells / mm 3

[0714] (3) HIV-1 viral load > 5,000 copies / mL

[0715] (4) no active infections requiring immediate treatment (other than HIV-1)

[0716] (5) no use of immunomodulatory drugs or systemic chemotherapy

[0717] (6) no need for highly active antiretroviral therapy (HAART).

[0718] Subjects follow the routine monitoring schedule (off antiretroviral drugs) or receive standard-of-care antiretroviral therapy (e.g., HAART) after the trial is completed when deemed necessary by the trial overall principal investigator in accordance with current guidelines for the diagnosis and treatment of HIV / AIDS. Individuals enrolled in the Phase I trial using UB-421 and individuals meeting the entry criteria for the Phase Ila trial are allowed to participate in this trial.

[0719] 4. Study Drug

[0720] UB-421 (dB4C7 mAb) is supplied at a concentration of 10 mg / mL (100 mg in a 10 mL vial).

[0721] Each enrolled subject received multiple intravenous infusions of UB-421 utilizing one of the following dose levels: 10 mg / kg weekly (Dose Cohort 1) or 25 mg / kg every two weeks (Dose Cohort 2) for a period of 8 weeks. The appropriate volume of UB-421 was based on the specific dose and subject body weight. The volume of each individual dose was adjusted using sterile normal saline, whereby an equivalent infusion volume of drug was infused for each individual subject within a dose cohort. The total infusion volume was approximately 100 mL for the 10 mg / kg dose cohort and approximately 200 mL for the 25 mg / kg dose cohort. The infusion time for each administration was approximately one to two hours.

[0722] 5. Assessment Criteria:

[0723] 5.1. Primary Safety and Efficacy Endpoints:

[0724] The following safety and tolerability parameters of UB-421 were assessed until Week 16 (end of trial):

[0725] (1) Physical Examination (PE)

[0726] (2) Vital Signs

[0727] (3) Clinical Chemistry and Hematology Tests

[0728] (4) Incidence of adverse events (AEs) / serious adverse events (SAEs)

[0729] The following efficacy parameters of UB-421 were assessed during the trial period (from V2 to V12) for each dose cohort:

[0730] (1) Individual maximum viral load reduction

[0731] (2) Mean maximum viral load reduction

[0732] 5.2. Secondary Virology Endpoints

[0733] The following virological responses were assessed during the trial period (from V2 to V12):

[0734] (1) Individual maximum viral load reduction and mean maximum viral load reduction according to subgroups within and between each trial dose cohort.

[0735] (2) Proportion of subjects with viral load < 50 copies / mL;

[0736] (3) Proportion of subjects with viral load < 200 copies / mL;

[0737] (4) Proportion of subjects with viral load reduction > 0.5 log 10 copies / mL;

[0738] (5) the proportion of subjects with > 1 log reduction in viral load 10 from baseline;

[0739] (6) the proportion of subjects with viral rebound (an increase of > 0.5 log 10 from nadir) in the 1st dose group and the 2nd dose group, respectively, up to 7 days and 14 days after the last administration of the test drug;

[0740] (7) serum concentration of anti-UB-421 antibody (immunogenicity of UB-421);

[0741] (8) changes in the number of CD4+ and CD8+ T cells;

[0742] (9) pharmacokinetic parameters (C max , AUC (0→∞) and AUC (0→last) ) of UB-421.

[0743] 6. Analysis Population

[0744] Intention-to-treat (ITT) population: 29 subjects who received at least one administration of the test drug. The ITT population of the 1st dose group and the 2nd dose group was 14 subjects and 15 subjects, respectively.

[0745] Per-protocol (PP) population: 18 subjects who received all administrations of the test drug, had valid baseline and at least one valid post-treatment efficacy measurement (HIV-1 viral load test), and had no major protocol violations. The PP population of the 1st dose group and the 2nd dose group was 7 subjects and 11 subjects, respectively.

[0746] Safety and immunogenicity population: 29 subjects were included in the intention-to-treat (ITT) population.

[0747] Pharmacokinetic population: was a subpopulation in the safety and immunogenicity population.

[0748] Baseline data and safety endpoints were analyzed in the safety and immunogenicity population, whereas efficacy analyses were conducted in both the ITT and PP populations. Pharmacokinetic analyses were conducted in the pharmacokinetic population.

[0749] 7. Duration of Trial Period

[0750] Screening period: < 4 weeks

[0751] Treatment period: 8 weeks

[0752] Follow-up period: 8 weeks following the end of the treatment period

[0753] Visit 0 represents the initial screening, while each visit during the trial represents a period of 1 week. The follow-up period is usually performed at weekly intervals.

[0754] 8. Summary of Results

[0755] 8.1. Trial Population

[0756] A total of 33 asymptomatic HIV-infected adults were screened at two trial sites in Taiwan. Of these, 29 subjects passed the screening criteria and were selected for the trial. All 29 eligible subjects were male.

[0757] 8.2. Safety and Tolerability Results

[0758] All 29 subjects experienced at least one adverse event during the trial, for a total of 128 adverse events. Of these, 114 (89.06% of all 29 subjects) were treatment-related adverse events (TEAEs) and 14 (10.94% of 5 subjects) were pre-treatment adverse events. No serious adverse events (SAEs) were observed in the 29 subjects. All pre-treatment adverse events were not related to UB-421, and none of these events were considered SAEs. The majority of reported TEAEs (78.95%) were mild, 17.54% were moderate, and 3.51% (in one subject) were severe.

[0759] The most common (>10%) TEAEs were rash and urticaria. In addition to adverse events, laboratory abnormality test results were observed in 22 subjects in hematology (154 events in 22 subjects) and biochemistry (32 events in 6 subjects). However, most of the changes were mild and not clinically significant. Physiological examination results and vital signs were mostly normal or not clinically significant during the trial period.

[0760] As stated in the clinical trial protocol, UB-421 was well-tolerated during the trial period, with an overall treatment tolerability of 73.84% for the 8-week treatment period.

[0761] 8.3. Pharmacodynamics

[0762] 8.3.1 CD4 + T and CD8 + T cell counts

[0763] After the 8-week treatment period and the 8-week follow-up period, the mean CD4 +T cell counts decreased slightly from baseline by 55.10 ± 117.97 cells / mm 3 and average CD8 + T cell counts increased from baseline by 193.31 ± 459.34 cells / mm 3 . Representative CD4 + T cell counts and average CD4 T cell counts for subjects in the 1st dose group are shown in Table 22A and FIG. 24A Representative CD4 + T cell counts and average CD4 T cell counts for subjects in the 2nd dose group are shown in Table 22B and FIG. 24B .

[0764] 8.3.2 CD4 Receptor Coverage with UB-421

[0765] The degree of CD4 receptor coverage was measured by flow cytometry using fluorescently conjugated UB-421. Results from four representative subjects, two from the 1st dose group and two from the 2nd dose group, are shown in FIG. 25A to 25B and FIG. 25C to 25D , respectively. The sensitivity of this assay was 0.15 μg / mL. The clinical efficacy of UB-421 was shown to reduce virus to below the limit of detection in the presence of UB-421 serum levels > 10 μg / mL when given as a monotherapy at 10 mg / kg weekly or 25 mg / kg every two weeks. There was no viral rebound as long as PBMC CD4+ cells were fully covered (i.e., dB4C7-Alexa binding percentage close to 0).

[0766] Full coverage of CD4 receptors on PBMC with UB-421 was achieved after two to three doses at both dose levels. In addition, full coverage of CD4+ T cells with UB-421 was maintained throughout the treatment period FIG. 25A - 25D, top panel). In most subjects, UB-421 binding to CD4 receptors decreased and returned to baseline values within three weeks of the final UB-421 infusion as determined by binding of the fluorescent dB4C7 mAb (dB4C7-Alexa).

[0767] The concentration of UB-421 present in the serum of subjects during the trial was evaluated to determine the serum concentration of UB-421 sufficient to achieve full CD4 coverage and HIV-1 viral suppression. Based on the data obtained, sustained full coverage of CD4+ T cells and HIV-1 viral suppression with UB-421 was achieved as long as UB-421 serum concentrations were maintained above 10 μg / mL FIG. 25A - 25D, bottom panel).

[0768] 8.4. Pharmacokinetics

[0769] The mean AUC was observed to increase from 17300 ± 10000 pg x hour / mL (Visit 1-2) to 23900 ± 10700 pg x hour / mL (Visit 8-9) and then returned to baseline at Visit 11-12 in the 1stdose cohort. The mean AUC observed in the 1stdose cohort (0→last) was 171000 ± 70300 pg x hour / mL.

[0770] The mean AUC was observed to increase from 56500 ± 19500 pg x hour / mL (Visit 1-3) to 61100 ± 20700 pg x hour / mL (Visit 7-9) and then returned to baseline at Visit 11-12 in the 2nddose cohort. The mean AUC observed in the 2nddose cohort (0→last) was 239000 ± 73900 pg x hour / mL.

[0771] These data demonstrate that, as measured by AUC (0→last) , the mean serum drug concentration was higher in subjects administered a 25 mg / kg UB-421 infusion every two weeks (2nddose cohort, 239000 ± 73900 pg x hour / mL) compared to subjects receiving a 10 mg / kg UB-421 infusion weekly (1stdose cohort, 171000 ± 70300 pg x hour / mL).

[0772] 8.5. Efficacy Results

[0773] This trial enrolled 29 HIV-1 infected subjects who received at least one dose of UB-421 (ITT population). Of the 29 subjects enrolled, a total of 18 subjects completed the 8-week treatment period and received all dosings of the trial medication (PP population). The efficacy of multiple doses of UB-421 was assessed by evaluating the individual and mean maximum viral load reduction in asymptomatic HIV-1 infected subjects enrolled in the trial during the trial period, and the results for the ITT and PP populations for the 1stand 2nddose cohorts are summarized in Table 20.

[0774] The mean maximum viral load reduction was found to be not significantly different between the two dose levels in either the ITT or PP population. Specifically, the viral load was reduced by 2.27 ± 0.60 log 10 copies / mL in the ITT population in the 1stdose cohort, and by 2.45 ± 0.46 log10 viral load reduction of 2.73 ± 0.34 log copies / mL in the PP population, while in the second dose group, viral load reduction was 2.47 ± 0.45 log copies / mL. 10 10

[0775] During the treatment period, viral load reduction of > 0.5 log copies / mL was observed in all (n=29, 100.00%) trial subjects; and viral load reduction of > 1 log copies / mL was observed in all (n=29, 100.00%) trial subjects. 10 10 During the treatment period, viral load reduction of > 0.5 log copies / mL was observed in all (n=29, 100.00%) trial subjects; and viral load reduction of > 1 log copies / mL was observed in all (n=29, 100.00%) trial subjects.

[0776] Further evaluation of data obtained during the treatment period is disclosed below:

[0777] In the first dose group, 8 / 14 (57.14%) subjects in the ITT and 5 / 7 (71.43%) subjects in the PP had viral load of < 200 copies / mL; in addition, 3 / 14 (21.43%) subjects in the ITT and 3 / 7 (42.86%) subjects in the PP had viral load of < 50 copies / mL.

[0778] In the second dose group, 10 / 15 (66.67%) subjects in the ITT and 7 / 11 (63.64%) subjects in the PP had viral load of < 200 copies / mL; and 3 / 15 (20.00%) subjects in the ITT and 2 / 11 (18.18%) subjects in the PP had viral load of < 50 copies / mL.

[0779] Viral load reduction data from representative subjects in the first and second dose groups are shown in Tables 21A to 21C and FIG. 25A to 25D (top panel). There were no statistically significant differences in the proportion of subjects with viral load reduction within each dose group, between each dose group, or between subpopulations within each dose group. In addition, during the 8-week treatment period, viral load decreased to levels below the current assay limit of detection (20 copies / mL) in 43% and 18% of subjects in the first and second dose groups, respectively. In all subjects, viral load was consistently reduced when CD4+ T cells were fully covered with UB-421. At the end of the follow-up period, viral load returned to baseline levels in both dose groups. In addition, no viral rebound was observed in any of the trial subjects during the treatment period. No measurable anti-UB421 antibodies were detected from patients in both dose groups throughout the treatment period (Tables 21A to 21C).

[0780] ​​​8.6. Comparison of UB-421 and TMB-355

[0781] Results provided by similar trials performed by others (Jacobson, J.M., et al., 2009; Toma, J., et al., 2011; and Pace, C.S., et al., 2013) with TMB-355 (ibalizumab, formerly known as TNX-355) were used to assess the results provided by UB-421 in this trial. FIG. 26A showed a high >3 Log 10 viral load reduction without viral load rebound in the presence of complete CD4+ cell coverage. In contrast, patients receiving TMB-355 treatment experienced viral rebound, a sign of development of resistant viral mutants, as early as one week after treatment, even in the presence of complete CD4+ cell coverage FIG. 26B .

[0782] The comparison of these two treatment regimens, as shown in the figure, demonstrates the clear advantage of using UB-421 to treat HIV-infected subjects over TMB-355 treatment. Specifically, UB-421 provided a sustained reduction in HIV viral load throughout the treatment period, and even one or two weeks into the follow-up period had a maximum viral load reduction of >3 Log 10 . In contrast, TMB-355 provided only a temporary viral load reduction by the first dose, and a maximum viral load reduction of close to 1 Log 10 .

[0783] Furthermore, previous trials using TMB-355 found that HIV viral rebound occurred as early as one week after treatment, despite the presence of serum TMB-355 and complete coverage of CD4-positive T cells (Jacobson, J.M., et al., 2009). This result is consistent with the previous prediction in Example 4 above that a non-competitive entry inhibition mechanism, such as that mediated by TMB-355 (ibalizumab), can provide a high likelihood of development of resistant HIV mutants during the antibody treatment period. Indeed, viral resistant mutants were found to discriminate mutations in the V5 region of gpl20 from patients receiving TMB-355 treatment to reduce viral load (Toma, J., et al., 2011; Pace, C.S., et al., 2013).

[0784] 9. Conclusion

[0785] Treatment of asymptomatic HIV-1 infected subjects with UB-421 for 8 weeks was well tolerated. In addition, the number of CD4+ T cells in individual subjects, represented by the median CD4 T cell counts (Tables 22A and 22B) and the mean CD4 T cell counts FIG. 24A and 24B remained stable throughout the two-month monitoring period.

[0786] More importantly, treatment with UB-421 resulted in a significant viral load reduction in all subjects (100% of treated subjects responded with a maximum reduction of > 1 log 10 copies / mL). Both treatment regimens, 10 mg / kg weekly (dose group 1) and 25 mg / kg biweekly (dose group 2) infusions, showed similar efficacy in viral load reduction. The mean maximum viral load reduction in the ITT population reached 2.27 ± 0.60 log 10 copies / mL in dose group 1 and 2.45 ± 0.46 log 10 copies / mL in dose group 2. The observed viral reduction efficacy with UB-421 is superior to any other small molecule anti-HIV drug tested to date.

[0787] The clinical trial results obtained from this carefully conducted multi-dose phase Ila of UB-421 demonstrate high tolerability, safety as monotherapy and unprecedented efficacy in viral load reduction without viral rebound during the treatment period. The results obtained in this trial are unexpected and contradict long-held skepticism in the field that anti-CD4 monoclonal antibodies binding to CD4 domain 1 are immunosuppressive due to their interference with second class major histocompatibility complex-mediated immune functions and that such therapy is not suitable for the treatment of HIV disease (Jacobson, J.M., et al., 2009). This result further points to additional modalities of HIV therapy with UB-421 in combination with orthogonal HAART and / or other HIV viral reservoir activators (e.g. HDACi) that can achieve functional cure of HIV infection.

[0788] Example 15. Treatment modalities with UB-421 monotherapy as a replacement for antiretroviral therapy in HIV-1 infected adults

[0789] FIG. 27 Treatment modalities with UB-421 monotherapy as a replacement for antiretroviral therapy in various HIV patient populations are illustrated. Detailed objectives and protocols are described below.

[0790] 1. Applicable patient population

[0791] Subjects who are seropositive for HIV-1 with viral suppression by stable highly active antiretroviral therapy (HAART) are eligible for this treatment.

[0792] Eligible patients receive UB-421 via IV or SC route for a 4-month initial period, followed by another cycle of HAART treatment. The "HAART-UB-421" alternating treatment cycle can be repeated multiple times until viral rebound is no longer observed upon discontinuation of UB-421 and HAART treatment, thereby achieving functional cure of HIV infection.

[0793] More specifically, these subjects receive multiple intravenous infusions of the study drug (UB-421) at one of two dose levels (10 mg / kg weekly or 25 mg / kg every two weeks) during treatment periods of 8 and 16 weeks, respectively. HAART treatment is discontinued one day prior to the first UB-421 infusion. Prior to UB-421 administration, subjects are given premedication (pre-treatment medication) comprising steroids and antihistamines, as judged by the overall trial director, to prevent infusion reactions. Upon completion of the last scheduled UB-421 administration, all subjects restart their previous or other appropriate viral sensitive antiretroviral therapy on the same day. The use of HAART treatment regimen is at the discretion of the overall trial director. Viral load and CD4 cell counts are monitored from all patients during the treatment period and for 6 months after the end of the treatment period.

[0794] 2. Inclusion criteria

[0795] A subject is included in this treatment modality if he / she meets all of the following criteria:

[0796] (1) HIV seropositive;

[0797] (2) Age 20 years or older;

[0798] (3) Has received HAART treatment defined as at least 2 nucleoside / nucleotide reverse transcriptase inhibitors (NRTIs) plus 1 non-nucleoside reverse transcriptase inhibitor (NNRTI), integrase inhibitor, or protease inhibitor for at least 2 years; this treatment was uninterrupted and without drug change within one year prior to entering the trial.

[0799] (4) Has had two measurements of CD4+ T cell count > 500 cells / mm 3 or CD4 percentage > 28% within 1 year prior to the screening visit;

[0800] (5) CD4+ T cell count < 200 cells / mm3within 4 weeks prior to the Screening Visit or at the Screening Visit 3 ;

[0801] (6) HIV-1 plasma RNA has remained below the limit of detection for at least 1 year prior to the Screening Visit, with at least 2 viral load determinations per year. Viral load was also below the limit of detection within 4 weeks prior to the Screening Visit or at the Screening Visit; a single episode of detectable HIV plasma RNA more than 4 weeks prior to the Screening Visit will not exclude participation.

[0802] 3. Exclusion Criteria

[0803] A subject can be excluded from this treatment for any of the following reasons:

[0804] (1) Any infection (except HIV) requiring immediate treatment;

[0805] (2) Any prior diagnosis or current AIDS-defining illness according to the United States Centers for Disease Control and Prevention (CDC) classification system for HIV infection under Category B and Category C conditions;

[0806] (3) Body weight > 80 kg;

[0807] (4) Any documented CD4+ T cell count < 250 cells / mm3or CD4+ T cell percentage < 14% within 12 weeks prior to screening; 3

[0808] (5) Prior inclusion in Phase I or Phase Ila trials of UB-421, or any history of anti-UB-421 antibodies;

[0809] (6) Any prior exposure to monoclonal antibodies within 12 weeks prior to the first dose of study drug UB-421;

[0810] (7) Any significant illness (except HIV-1 infection) or clinically significant findings, including psychiatric and behavioral problems, determined by screening, medical history, and / or physical examination, which in the judgment of the Investigator exclude the subject from participation in the trial;

[0811] (8) Any vaccination within 8 weeks prior to the first dose of study drug;

[0812] (9) Any immunomodulatory treatment (including interferon), systemic chemotherapy within 12 weeks prior to the first dose of study drug;

[0813] (10) Life expectancy of less than 12 months;

[0814] ​(11) Any non-medical intravenous drug use within 12 weeks prior to the first dose of study drug;

[0815] (12) More than one change in HAART regimen due to virologic failure and prior non-Hodgkin's lymphoma or Kaposi's sarcoma;

[0816] (13) Any current alcohol or non-medical drug use that, in the judgment of the study director, would interfere with the ability of the subject to comply with the dosing and visit schedule and with the ability to perform the assessments of the protocol.

[0817] 4. Drug Product

[0818] Drug product UB-421 (dB4C7 mAb) was supplied at a concentration of 10 mg / mL (100 mg in a 10 mL vial). Subjects received 8 weekly doses of 10 mg / kg UB-421 or 8 biweekly doses of 25 mg / kg UB-421 as an intravenous infusion.

[0819] The appropriate volume of UB-421 was based on the specific dose and the subject's body weight. The volume of each individual dose was adjusted with sterile normal saline, whereby each individual subject within a dose group was infused with an equivalent volume of drug infusion. The total volume of infusion for the 10 mg / kg dose group was approximately 100 mL, while the total volume of infusion for the 25 mg / kg dose group was approximately 200 mL. The infusion time for each dose was approximately one to two hours.

[0820] Example 16. Treatment modalities for functional cure of HIV-1 infection using UB-421 in combination with HAART

[0821] FIG. 28 It is demonstrated that treatment modalities using UB-421 in combination with HAART can provide for a functional cure of HIV-1 infection in various patient populations. The applicable patient populations are: (1) HIV patients not using therapeutic drugs; (2) HIV patients stable on HAART therapy; and (3) HIV patients with HAART therapy failure.

[0822] More specifically, the achievement of a clinical protocol for the functional cure of HIV in infected subjects is by the administration of UB-421 through the IV or SC route in the initial phase (4 months), followed by a treatment holiday (2 months) as one treatment cycle (6 months) in two complete cycles (1 year). These same subjects also start and continue HAART treatment during the two complete cycles of UB-421 treatment. At the end of the two complete cycles, HAART and UB-421 are discontinued (Arm A) to assess the time required for a viral rebound, if any. In the control group, subjects treated with HAART alone during the same 12-month period before HAART treatment is discontinued are also assessed to determine the time of viral rebound, if any (Arm B).

[0823] Viral load and CD4 cell counts from all patients are monitored during the two cycles of UB-421 and HAART treatment and 6 months after treatment, for a total of 18 months.

[0824] Arm A: treatment with HAART combined with UB-421 administered at 10 mg / kg weekly or 25 mg / kg every two weeks. Arm B: treatment with HAART alone.

[0825] 1. Design of UB-421 treatment for functional cure (Table 23)

[0826] Potential advantages of UB-421 over HAART drugs:

[0827] (1) UB-421 blocks the intercellular spread of HIV-1 virus

[0828] (2) UB-421 crosslinks the CDR-2-like loop of CD4 and activates the cell, and thus induces and releases HIV-1 from latency

[0829] 2. Objectives

[0830] (1) To provide effective treatment and protection for HIV-infected subjects by blocking the cell-free and intercellular spread of HIV using UB-421 in combination with HAART.

[0831] (2) To provide a functional cure for HIV in HIV-infected subjects.

[0832] 3. Applicable patient population

[0833] (1) HIV patients on stable HAART treatment; (2) HIV patients not treated with HAART; and (3) HIV patients who have failed HAART treatment.

[0834] 4. Drug Substance UB-421

[0835] Drug Substance UB-421 (dB4C7 mAb) is provided at a concentration of 10 mg / mL (100 mg in a 10 mL vial). Subjects receive 8 weekly doses of 10 mg / kg UB-421 or 8 biweekly doses of 25 mg / kg UB-421 by intravenous infusion.

[0836] The appropriate volume of UB-421 is based on the specific dose and the subject's body weight. The volume of each individual dose is adjusted using sterile normal saline, whereby each individual subject within a dose group is infused with an equivalent volume of drug infusion. The total volume of infusion for the 10 mg / kg dose group is approximately 100 mL, while the total volume of infusion for the 25 mg / kg dose group is approximately 200 mL. The infusion time for each administration is approximately one to two hours.

[0837] 5. Allocation Intervention

[0838] The following intervention is allocated:

[0839] 5.1. Group A - Combination of UB-421 and HAART therapy

[0840] Subjects are treated continuously with an appropriate HAART regimen and are also treated with UB-421 for two complete cycles of one year. Each cycle of UB-421 treatment consists of 10 mg / kg UB-421 administered weekly for a period of 4 months or 25 mg / kg UB-421 administered biweekly, followed by two months of no UB-421 treatment.

[0841] At the end of the one year treatment period, HAART and UB-421 treatment are discontinued. Additional observation is conducted (by virtue of the lack of any viral rebound in the absence of HAART and UB-421) to ensure functional cure of HIV infection.

[0842] 5.2. Group B - HAART therapy alone

[0843] As a control group, a separate group of subjects is treated continuously with an appropriate HAART regimen without UB-421 treatment for the same period.

[0844] At the end of the one year treatment period, HAART treatment is discontinued and subjects are monitored for viral rebound.

[0845] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed in this specification is only an example of a generic series of similar features.

[0846] From the above, it is manifest that this application can be readily utilized in any particular environment with the necessary features, and various modifications and alterations to this application can be made to adapt the application to various usages and conditions, as will be apparent to those with ordinary skill in the art, without departing from the spirit and scope of the application.

[0847] Table 1. Unmet medical need in HIV treatment: functional cure and eradication

[0848]

[0849] Table 2. List of HDAC inhibitors in development for reactivation of HIV-infected silent T cells

[0850]

[0851]

[0852] Table 3. HIV entry inhibition activity of monoclonal antibody B4 (Monogram Biosciences PhenoSense™ assay)

[0853]

[0854] Table 4. Corresponding CDR 1, 2, 3 sequences from murine antibody B4 heavy and light chain sequences

[0855]

[0856] Table 5. Neutralization activity of deimmunized B4 (dB4C7) compared to parental B4 (MT-2 microbplaque assay)

[0857]

[0858] Table 6. Neutralization activity of deimmunized B4 (dB4C7) compared to parental B4 (PBMC assay)

[0859]

[0860]

[0861] Table 8. Binding affinity of dB4C7 to HPB-ALL cells (EC 50 )

[0862]

[0863] Table 9. Absolute binding affinity (Kd) and maximum binding capacity (Bmax) of dB4C7 to HPB-ALL cells

[0864]

[0865] Table 10. Binding activity (EC50) of dB4C7 to blood CD4+ T cells measured using (1) goat anti-huIgG-FITC and (2) dB4C7-Alexa binding 50 )

[0866]

[0867] Table 11. Monoclonal antibody B4 blocks HIV cell-free and cell-to-cell transmission

[0868]

[0869] Table 12. Sequential staining with FACS analysis - percentage of positive PBMC

[0870]

[0871] Table 13. TNF-α levels and HIV-1 viral load in PBMC cultures

[0872]

[0873] ND: below detection limit

[0874] Table 14A. Experiment A: single administration in baboons preclinical trial

[0875]

[0876] Table 14B. Experiment B: multiple administrations in baboons preclinical trial

[0877]

[0878] 1 8-week period every week

[0879] 2 Dose: mg (UB-421) / kg (body weight)

[0880] Table 15. Blood and biopsy results in baboons

[0881]

[0882] Table 16. Observations and necropsy results in baboons

[0883]

[0884] Table 17. Preclinical Overview of UB-421: IND-Enabling Toxicology Studies in Baboons

[0885]

[0886] Table 18. Assessment of Cross-reactivity of Tissues Using Biotinylated mAb dB4C7

[0887]

[0888] 1 Strongly positive surface membrane staining was observed

[0889] 2 Pre-incubation with unlabelled mAb dB4C7 blocked staining

[0890] 3 Weakly positive was observed

[0891] 4 Occasional positive staining was observed

[0892] 5 Focal weak Kuppfer cell staining

[0893] Table 19. PK Parameters for Single Dose UB-421 in Phase I Trial

[0894]

[0895] 1 Results are the mean from 3 subjects

[0896] Table 20. Viral Load Reduction Following Multiple Doses of UB-421 in Phase Ila Trial

[0897]

[0898] ITT: Intent-to-Treat Population

[0899] PP: Per-Protocol Population

[0900] VL: Viral Load

[0901] Table 21A. Phase Ila Clinical Efficacy Data Showing Viral Load Reduction to Below the Limit of Detection: Patient 1-1-01 (Dose Group 1: 10 mg / kg weekly) 1 Strongly positive surface membrane staining was observed

[0889] 2 Pre-incubation with unlabelled mAb dB4C7 blocked staining

[0890] 3 Weakly positive was observed

[0891] 4 Occasional positive staining was observed

[0892] 5 Focal weak Kuppfer cell staining

[0893] Table 19. PK Parameters for Single Dose UB-421 in Phase I Trial

[0894]

[0895] 1 Results are the mean from 3 subjects

[0896] Table 20. Viral Load Reduction Following Multiple Doses of UB-421 in Phase Ila Trial

[0897]

[0898] ITT: Intent-to-Treat Population

[0899] PP: Per-Protocol Population

[0900] VL: Viral Load

[0901] Table 21A. Phase Ila Clinical Efficacy Data Showing Viral Load Reduction to Below the Limit of Detection: Patient 1-1-01 (Dose Group 1: 10 mg / kg weekly) 1 Strongly positive surface membrane staining was observed

[0889] 2 Pre-incubation with unlabelled mAb dB4C7 blocked staining

[0890] 3 Weakly positive was observed

[0891] 4 Occasional positive staining was observed

[0892] 5 Focal weak Kuppfer cell staining

[0893] Table 19. PK Parameters for Single Dose UB-421 in Phase I Trial

[0894]

[0895] 1 Results are the mean from 3 subjects

[0896] Table 20. Viral Load Reduction Following Multiple Doses of UB-421 in Phase Ila Trial

[0902]

[0903] NQ: Not Quantifiable

[0904] / : Not Evaluated

[0905] Table 21B. Phase Ila clinical efficacy data showing viral load reduction to below the limit of detection: Patient 1-1-02 (Dose 1 group: 10 mg / kg weekly)

[0906]

[0907] NQ: Not Quantifiable

[0908] / : Not Evaluated

[0909] Table 21C. Phase Ila clinical efficacy data showing viral load reduction to below the limit of detection: Patient 1-2-03 (Dose 2 group: 25 mg / kg biweekly)

[0910]

[0911] NQ: Not Quantifiable

[0912] / : Not Evaluated

[0913] Table 22A. Mean CD4 T cell numbers for UB-421 treated patients in Dose 1 group (10 mg / kg weekly)

[0914]

[0915] Table 22B. Mean CD4 T cell numbers for UB-421 treated patients in Dose 2 group (25 mg / kg biweekly)

[0916]

[0917] Table 23. Design of UB-421 treatment for functional cure

[0918]

[0919] SEQUENCE LISTING <110>United Biomedical, Inc. Wang Changyi <120>Treatment and functional cure of HIV infection by competitive HIV entry inhibition mediated by monoclonal antibodies against CD4 <130>1004263.213WO (2034WO) <150>US 62 / 051,200 <151> 2014‑09‑16 <150>PCT / US2014 / 065048 <151> 2014‑11‑11 <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213>murine antibody <220> <221>domain <222> (1)..(5) <223>CDR1 of the heavy chain of murine antibody B4 <400> 1 Asp Tyr Val Ile His 1 5 <210> 2 <211> 17 <212> PRT <213>murine antibody <220> <221>domain <222> (1)..(17) <223>CDR2 of the heavy chain of murine antibody B4 <400> 2 Glu Ile Tyr Pro Gly Ser Gly Ser Ala Tyr Ser Asn Ala Lys Phe Lys 1 5 10 15 Asp <210> 3 <211> 9 <212> PRT <213>murine antibody <220> <221>domain <222> (1)..(9) <223>CDR3 of the heavy chain of murine antibody B4 <400> 3 Arg Gly Asn Gly Thr Gly Phe Ala Tyr 1 5 <210> 4 <211> 15 <212> PRT <213> murine antibody <220> <221> domain <222> (1)..(15) <223> CDR1 of light chain of murine antibody B4 <400> 4 Lys Ala Gly Gin Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> murine antibody <220> <221> domain <222> (1)..(7) <223> CDR2 of light chain of murine antibody B4 <400> 5 Val Ala Ser Asn Leu Glu Ser 1 5 <210> 6 <211> 9 <212> PRT <213> murine antibody <220> <221> domain <222> (1)..(9) <223> CDR3 of light chain of murine antibody B4 <400> 6 Gln Gin Ser Tyr Lys Asp Pro Leu Thr 1 5 <210> 7 <211> 448 <212> PRT <213> murine antibody <220> <221> domain <222> (1)..(448) <223> Heavy chain of de-immunized human antibody B4 [UB421] having CDRs 1, 2, 3 similar to CDRs 1, 2, 3 derived from CDRs 1, 2, 3 corresponding to the heavy chain sequence of the parental murine B4 antibody <400> 7 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Val Ile His Trp Val Lys Gln Ala Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ala Tyr Ser Asn Ala Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Gly Asn Gly Thr Gly Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr His Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 8 <211> 218 <212> PRT <213> Murine antibody <220> <221> Domain <222> (1)..(218) <223> Light chain of deimmunized human antibody B4 [UB421] having CDRs 1, 2, 3 similar to CDRs 1, 2, 3 derived from the CDRs 1, 2, 3 corresponding to the light chain sequence of the parental murine B4 antibody <400> 8 Asp Ile Val Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Lys Ala Gly Gin Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Val Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Asn Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Ser Tyr 85 90 95 Lys Asp Pro Leu Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Leu Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 9 <211> 448 <212> PRT <213> Murine antibody <220> <221> Domain <222> (1)..(448) <223> Heavy chain of de-immunized human antibody B4 [UB421] with M253Y / S255T / T257E substitutions <400> 9 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Val Ile His Trp Val Lys Gin Ala Thr Gly Gin Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ala Tyr Ser Asn Ala Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Gly Asn Gly Thr Gly Phe Ala Tyr Trp Gly Gin Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gin Thr Tyr He Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr He Thr Arg 245 250 255 Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gin Tyr His Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys Thr 325 330 335 He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 10 <211> 448 <212> PRT <213> Humanized antibody <220> <221> Unassigned feature <222> (253)..(253) <223> M or Y <220> <221> Unassigned feature <222> (255)..(255) <223> S or T <220> <221> Unassigned feature <222> (257)..(257) <223> T or E <220> Features not yet classified <222> (298)..(298) <223>N or H <400> 10 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Val Ile His Trp Val Lys Gln Ala Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ala Tyr Ser Asn Ala Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Gly Asn Gly Thr Gly Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Xaa Ile Xaa Arg 245 250 255 Xaa Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Xaa Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 11 <211> 118 <212> PRT <213> Murine antibody <400> 11 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Val Ile His Trp Val Lys Gln Ala Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ala Tyr Ser Asn Ala Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Gly Asn Gly Thr Gly Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 12 <211> 330 <212> PRT <213> Human antibody <220> <221> Unassigned feature <222> (135)..(135) <223> M or Y <220> <221> Unassigned feature <222> (137)..(137) <223> S or T <220> <221> Uncharacterized feature <222> (139)..(139) <223> T or E <220> <221> Uncharacterized feature <222> (180)..(180) <223> N or H <400> 12 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Xaa Ile Xaa Arg Xaa Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Xaa Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 13 <211> 111 <212> PRT <213> Murine antibody <400> 13 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Lys Ala Gly Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Val Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Asn Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr 85 90 95 Lys Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 14 <211> 107 <212> PRT <213> Human antibody <400> 14 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Leu Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105

Claims

1. Use of an antibody to CD4 domain 1 in the preparation of a medicament for treating human immunodeficiency virus (HIV), comprising administering to a subject exposed to HIV a pharmacologically effective amount of the antibody, and wherein the antibody is a humanized monoclonal antibody comprising a heavy chain of the amino acid sequence of SEQ ID NO: 7 or 9; and a light chain of the amino acid sequence of SEQ ID NO:

8.

2. The use of claim 1, wherein the antibody specifically binds to a CDR2 region located in CD4 domain 1.

3. The use of claim 1, wherein the humanized antibody is administered to the subject prior to exposure to HIV.

4. The use of claim 1, wherein the humanized antibody is administered to the subject after exposure to HIV.

5. The use of claim 4, wherein the humanized antibody is administered within 48 hours after exposure to HIV.

6. The use of claim 1, wherein the humanized antibody is administered to the subject at a dose of at least 5 mg / kg body weight.

7. The use of claim 6, wherein the humanized antibody is administered to the subject multiple times.

8. The use of claim 7, wherein the humanized antibody is administered to the subject at intervals of between every week or every two weeks.

9. The use of claim 7, further comprising administering to the subject an antiviral drug.

10. The use of claim 9, wherein the antiviral drug is highly active antiretroviral therapy (HAART).

11. The use of claim 10, wherein the HAART comprises a nucleoside analog reverse transcriptase inhibitor in combination with a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor.

12. The use of claim 10, wherein the humanized antibody is administered concurrently with the HAART.

13. The use of claim 10, wherein the humanized antibody and the HAART are administered to the subject for a cycle, wherein the cycle comprises: i) administering the humanized antibody to the subject at intervals of between every week or every two weeks for a period of 4 months, followed by a two month treatment holiday; and ii) administering HAART to the subject continuously during the six month period in (i).

14. The use of claim 13, wherein the subject is treated for two or more cycles.

15. Use of an antibody to CD4 domain 1 and highly active antiretroviral therapy (HAART) in the preparation of a medicament for treating HIV, comprising administering to a subject the medicament for treating HIV, the medicament for treating HIV comprising: a) a pharmacologically effective amount of an antibody to CD4 domain 1, wherein the antibody is a humanized monoclonal antibody comprising a heavy chain of the amino acid sequence of SEQ ID NO: 7 or 9; and a light chain of the amino acid sequence of SEQ ID NO: 8; and b) highly active antiretroviral therapy (HAART).

16. The use of claim 15, wherein the antibody specifically binds to a CDR2 region located in CD4 domain 1. ​ ​ ​ ​ 17. The use of claim 15, wherein the humanized antibody is administered to the subject at a dose of at least 5 mg / kg body weight.

18. The use of claim 15, wherein the subject is administered the therapeutic HIV drug for a cycle, wherein the cycle comprises: i) administering the humanized antibody to the subject for a period of four months at a weekly or biweekly interval, followed by a two month treatment holiday; and ii) administering HAART to the subject continuously during the six month period of (i).

19. The use of claim 18, wherein the subject is treated for two or more cycles.

20. An antibody against a CD4 molecule, wherein the antibody is a humanized monoclonal antibody comprising: a heavy chain of the amino acid sequence of SEQ ID NO: 7 or 9; and a light chain of the amino acid sequence of SEQ ID NO: 8, and the antibody: (a) binds to an extracellular region of the CD4 molecule; (b) competitively inhibits HIV entry into a cell; and (c) reduces HIV viral load in an HIV-positive patient to less than 50 copies / mL of blood without viral load rebound when a sufficient amount of the antibody is administered to the HIV-positive patient to completely coat CD4+ cells. The antibody activates quiescent CD4+ cells once cross-linking is formed.

22. The antibody of claim 20, wherein the antibody binds to a region near domain 1 of the CD4 molecule.

23. The antibody of claim 20, wherein the antibody binds to a region near the CDR2 region of domain 1 of CD4.

25. The antibody of claim 20, wherein the antibody binds to a CD4 molecule.

26. The antibody of claim 20, wherein the antibody binds to a CD4 molecule on the surface of a T cell.

21. The antibody of claim 20, wherein, 27. A composition comprising the antibody of claim 20.

28. A pharmaceutical composition comprising the antibody of claim 20 and a pharmaceutically acceptable carrier.

29. A pharmaceutical composition comprising the antibody of claim 20 dissolved in a phosphate buffer comprising 20 mM glycine and 0.05% (v / v) polysorbate 20.

24. The antibody of claim 20, wherein the antibody has an absolute binding affinity (Kd) for membrane-bound CD4 located on HPB-ALL cells of between 3.1 x 10 -11 M and 8.1 x 10 -11 M.

30. A pharmaceutical composition comprising the antibody of claim 20 dissolved in a phosphate buffer comprising 20 mM glycine, 0.05% (v / v) polysorbate 20, and 10 mM histidine.

31. A pharmaceutical composition comprising 1.0 mg / mL to 200.0 mg / mL of the antibody of claim 20 dissolved in a phosphate buffer comprising 20 mM glycine and 0.05% (v / v) polysorbate 20.

32. A pharmaceutical composition comprising 1.0 mg / mL to 200.0 mg / mL of the antibody of claim 20 dissolved in a phosphate buffer comprising 20 mM glycine, 0.05% (v / v) polysorbate 20, and 10 mM histidine. ​ ​ ​ ​ ​ 33. A pharmaceutical composition comprising 10.0 mg / mL of the antibody of claim 20 in a phosphate buffer comprising 20 mM glycine and 0.05% (v / v) polysorbate 20.

34. A pharmaceutical composition comprising 10.0 mg / mL of the antibody of claim 20 in a phosphate buffer comprising 20 mM glycine, 0.05% (v / v) polysorbate 20 and 10 mM histidine.

35. Use of the antibody of claim 20 for the manufacture of a medicament for treating HIV, comprising administering a pharmacologically effective amount of the antibody to a subject exposed to HIV.

36. The use of claim 35, wherein the antibody is administered to the subject prior to exposure to HIV.

37. The use of claim 35, wherein the antibody is administered to the subject after exposure to HIV.

38. The use of claim 35, wherein the antibody is administered within 48 hours after exposure to HIV.

39. The use of claim 35, wherein the antibody is administered to the subject at a dose of at least 5 mg / kg body weight.

40. The use of claim 39, wherein the antibody is administered to the subject in multiple doses.

41. The use of claim 40, wherein the antibody is administered to the subject at intervals of between weekly, biweekly or monthly.

42. The use of claim 40, further comprising administering to the subject an antiviral agent.

43. The use of claim 42, wherein the antiviral agent is highly active antiretroviral therapy (HAART).

44. The use of claim 43, wherein the HAART comprises a nucleoside analog reverse transcriptase inhibitor in combination with a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor.

45. The use of claim 43, wherein the antibody is administered concurrently with the HAART.

46. The use of claim 43, wherein the antibody and the HAART are administered to the subject in a cycle, wherein the cycle comprises: i) administering the antibody to the subject for a first period, followed by a treatment holiday as a second period; and ii) administering the HAART to the subject continuously during the first period and the second period of (i).

47. The use of claim 43, wherein the antibody and the HAART are administered to the subject in a cycle, wherein the cycle comprises: i) administering the antibody to the subject at intervals of between weekly, biweekly or monthly for a period of 4 months, followed by a two month treatment holiday; and ii) administering the HAART to the subject continuously during the six month period of (i).

48. The use of claim 46, wherein the subject is treated for a period of two cycles.

49. The use of claim 47, wherein the subject is treated for a period of two cycles.

50. The use of claim 43, wherein the antibody is administered at a different time than the HAART. ​ ​ ​ ​ 51. The use of claim 43, wherein the antibody and the HAART are administered to the subject for a cycle of periods, wherein the cycle comprises: i) administering the antibody to the subject for a first period, followed by a treatment holiday that is a second period; and ii) administering the HAART to the subject during the second period but not the first period.

52. The use of claim 51, wherein the antibody is administered at regular intervals during the first period.

53. The use of claim 51, wherein the antibody is administered at weekly, biweekly, or monthly intervals during the first period.

54. Use of an antibody of claim 20 and a highly active antiretroviral therapy (HAART) for the manufacture of a medicament for treating HIV, comprising administering the medicament for treating HIV to a subject infected with HIV, the medicament for treating HIV comprising: a) a pharmacologically effective amount of an antibody of claim 20; and b) a highly active antiretroviral therapy (HAART).

55. The use of claim 54, wherein the antibody is administered to the subject at a dose of at least 5 mg / kg body weight.

56. The use of claim 54, wherein the antibody and the HAART are administered to the subject for a cycle of periods, wherein the cycle comprises: i) administering the antibody to the subject for a first period, followed by a treatment holiday that is a second period; and ii) administering the HAART to the subject continuously during the first period and the second period of (i).

57. The use of claim 54, wherein the antibody and the HAART are administered to the subject for a cycle of periods, wherein the cycle comprises: i) administering the antibody to the subject at weekly, biweekly, or monthly intervals for a period of 4 months, followed by a treatment holiday of two months; and ii) administering the HAART to the subject continuously during the six-month period of (i).

58. The use of claim 56, wherein the subject is treated for a cycle of two or more cycles.

59. The use of claim 57, wherein the subject is treated for a cycle of two or more cycles.

60. The use of claim 57, wherein the antibody and the HAART are administered to the subject for a cycle of periods, wherein the cycle comprises: i) administering the antibody to the subject at weekly, biweekly, or monthly intervals for a period of 4 months, followed by a treatment holiday of two months; and ii) administering the HAART to the subject continuously during the six-month period of (i).

61. The use of claim 54, wherein the antibody of (a) and the HAART of (b) are not administered at the same time.

62. The use of claim 54, wherein the antibody of (a) and the HAART of (b) are administered to the subject for a cycle of periods, wherein the cycle comprises: i) administering the antibody to the subject for a first period, followed by a treatment holiday that is a second period; and ii) administering the HAART to the subject during the second period but not the first period. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 63. The use of claim 62, wherein the antibody is administered at regular intervals during the first period.

64. The use of claim 62, wherein the antibody is administered at intervals of between weekly, biweekly, or monthly during the first period.

65. Use of an antibody of claim 20 for the manufacture of a medicament for inhibiting entry of HIV into a CD4+ cell, comprising contacting the antibody with the cell.

66. Use of an antibody of claim 20 for the manufacture of a medicament for inhibiting binding of gpl20 of HIV to a CD4+ cell, comprising contacting the antibody with the cell.

67. Use of an antibody of claim 20 for the manufacture of a medicament for silencing CD4+ T cells in an HIV patient when cross-linking occurs, comprising contacting the antibody with the cell.

68. Use of an antibody of claim 20 for the manufacture of a medicament for activating HIV latent reservoirs when cross-linking occurs in silencing T cells, comprising contacting the antibody with the cell.

Images