HK1189501B - Antibody compositions and methods of use - Google Patents

Description

Antibody compositions and methods of use

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application nos. 61/387,735 and 61/387,725, filed on 29/2010, and 61/504,056, filed on 1/7/2011, which are all incorporated herein by reference in their entirety.

Reference to sequence Listing submitted in text File over EFS-WEB

The sequence listing was filed simultaneously with the specification via EFS-Web as an ASCII formatted text file with a file name of "p 4680r1us. txt", creation date 2011 year, 9 month, 15 days, having a size of 200,277 bytes. The sequence listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to anti-complex I and anti-gH antibodies and methods of use thereof.

Background

Human Cytomegalovirus (HCMV) is a type β herpesvirus, also known as human herpesvirus type 5 (HHV-5). There are other Cytomegalovirus (CMV) species that infect other mammals, such as murine CMV (mcmv), guinea pig CMV (gpcmv), simian CMV (sccmv), rhesus CMV (rhcmv), and chimpanzee CMV (ccmv). HCMV is a common herpesvirus infecting nearly 50% of the us population. HCMV infection is asymptomatic for the vast majority of infected human individuals. However, under both diseased and immunosuppressive conditions (e.g., HIV infection, drug-induced immunosuppression in transplant patients), HCMV reactivation or primary infection can cause a variety of clinical manifestations, such as mononucleosis, hepatitis, retinitis, pneumonia, blindness, and organ failure. Furthermore, in the context of pregnancy, acquisition of a primary CMV infection, while having little effect on the mother, can have serious clinical consequences in the developing fetus.

Congenital HCMV infections are particularly important because many children born by mothers who are infected during pregnancy can become infected in utero and develop devastating clinical illness. In the united states and europe, 126,000 women have primary HCMV infection during pregnancy, and about 40,000 of the infants born to these mothers have congenital infections. In the united states, 1 of 750 children have disability or develop disability due to HCMV infection, including: mental retardation, hearing loss, vision loss, organ defects, and growth defects. Congenital HCMV infection is the most common cause of infection of fetal abnormalities. After a pregnant woman has developed a primary infection, there is currently no approved treatment for preventing or treating embryonic infections. Therefore, there is a great need in the art to find compositions and methods to prevent congenital HCMV infection.

In 2005, Nigro and colleagues disclosed a study in which human CMV hyper-immunoglobulin (HIG) was administered to a expectant parturient with a primary HCMV infection (Nigro et al (2005) New Engl. J. Med.353: 1350-. In one group of studies (arm), only 1 of the 31 infants born by HCMV-infected mothers had illness, whereas 7/14 (50%) of children born untreated women had illness from HCMV. As previously cited.

During pregnancy, HCMV can be transmitted from an infected mother to a fetus via the placenta. The placenta, which immobilizes the fetus to the uterus, contains specialized epithelial cells, stromal fibroblasts, endothelial cells, and specialized macrophages. The HCMV virus surface contains a variety of viral glycoprotein complexes that have been shown to be necessary to infect specific cell types in the placenta. The CMV glycoprotein complex containing gH/gL and UL128, UL130 and UL131 (referred to herein as "complex I") is particularly desirable for infection of endothelial cells, epithelial cells and macrophages. The CMV glycoprotein complex containing gH/gL and gO (referred to herein as "complex II") is particularly desirable for infection of fibroblasts. HIG has been shown to block viral entry into all four placental cells susceptible to HCMV infection.

Because HIG is difficult to prepare and widely distributed and doctors and medical groups are reluctant to use human blood products, particularly in pregnant women, it would be most advantageous to produce compositions comprising one or more monoclonal antibodies that can protect a fetus from congenital HCMV infection. To date, monoclonal antibody compositions for preventing maternal-infant transmission of CMV have not been developed. Lanzavecchia and Macagno have disclosed naturally occurring antibodies isolated from immortalized B cells of infected patients that bind to a conformational epitope caused by the combination of UL130 and UL131 or the combination of UL128, UL130 and UL131, neutralizing CMV transmission (U.S. patent publication nos. 2008/0213265 and 2009/0081230). Shenk and Wang have disclosed antibodies that bind to the protein of complex I (U.S. patent No. 7,704,510). Funaro et al also disclose neutralizing antibodies against CMV in U.S. patent publication No. 2010-0040602. In addition, the anti-gH monoclonal antibody MSL-109 was tested in humans, but was unsuccessful, in two patient populations, allogeneic bone marrow transplant recipients and patients with AIDS and CMV retinitis (Drobyski et al, Transplantation51: 1190-.

There remains a need in the art to develop monoclonal antibodies for preventing HCMV infection, including congenital HCMV infection.

Summary of The Invention

The invention provides isolated antibodies that specifically bind to HCMV complex I. In particular embodiments, an anti-complex I antibody of the invention comprises six HVRs (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 8; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 9; (e) HVR-L2 comprising an amino acid sequence selected from SEQ ID NOs: 10-19; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20. The antibody may further comprise the light chain variable domain framework FR1 comprising the amino acid sequence of SEQ ID NO 43 and FR2 comprising the amino acid sequence of SEQ ID NO 44.

In further embodiments, an anti-complex I antibody of the invention comprises three heavy chain hypervariable regions (HVR-H1, HVR-H2, and HVR-H3) and three light chain hypervariable regions (HVR-L1, HVR-L2, and HVR-L3), wherein (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO 6; (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO. 7; (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO. 8; (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO. 9; (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO. 20; and (e) the HVR-L2 and the first amino acid of the light chain variable domain framework FR3 comprise the amino acid sequence of SEQ ID NO: 21.

In particular embodiments, the anti-complex I antibody comprises (a) a VH comprising the amino acid sequence of SEQ ID NO:45, or SEQ ID NO:46, or SEQ ID NO: 47; and (b) a VL comprising the amino acid sequence of SEQ ID NO:48 or SEQ ID NO: 49. Such antibodies may further comprise the light chain variable domain framework FR3 comprising the amino acid sequence of SEQ ID NO:41 and FR4 comprising the amino acid sequence of SEQ ID NO: 42.

In some embodiments, the anti-complex I antibody comprises a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO 45, or SEQ ID NO 46, or SEQ ID NO 47, and a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO 48 or SEQ ID NO 49. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO 45, or SEQ ID NO 46, or SEQ ID NO 47. In some embodiments, the anti-complex I antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:48 or SEQ ID NO: 49. In some embodiments, the anti-complex I antibody comprises the VH sequence of SEQ ID NO 45 or SEQ ID NO 46 and the VL sequence of SEQ ID NO 49.

The invention also provides isolated antibodies that specifically bind to HCMV gH.

In some embodiments, an anti-gH antibody of the invention comprises three heavy chain hypervariable regions (HVR-H1, HVR-H2, and HVR-H3) and three light chain hypervariable regions (HVR-L1, HVR-L2, and HVR-L3), wherein:

(a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 71;

(b) HVR-H2 comprises an amino acid sequence selected from SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO: 93;

(c) HVR-H3 comprises the amino acid sequence of SEQ ID NO. 75;

(d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 76;

(e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 77; and

(f) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 78.

In some embodiments, the anti-gH antibody comprises HVR-H2 comprising the amino acid sequence of SEQ ID NO:93, wherein the amino acid at position 6 of SEQ ID NO:93 is selected from Ser, Thr, Asn, Gln, Phe, Met, and Leu, and the amino acid at position 8 of SEQ ID NO:93 is selected from Thr and Arg.

In particular embodiments, the anti-gH antibody comprises HVR-H2 comprising the amino acid sequence of SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO: 74.

In other embodiments, the anti-gH antibody comprises HVR-H2 comprising the amino acid sequence of SEQ ID NO:94, wherein the sequence comprises an amino acid selected from Ser, Thr, Asn, Gln, Phe, Met, and Leu at position 54 (of SEQ ID NO: 94). In some embodiments, the antibody further comprises an amino acid at position 56 selected from Thr and Arg.

The invention also provides an anti-gH antibody having a VH sequence which is at least 95% identical in amino acid sequence to SEQ ID NO 94, wherein the sequence comprises amino acids Asn54, Ser54, Thr54, gin 54, Phe54, Met54 or Leu54 and/or Arg 56. In particular embodiments, the antibody comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO: 89. In some embodiments, the VH comprises an amino acid sequence 95% identical to SEQ ID NO 94, wherein said sequence contains an amino acid at position 54 selected from Asn54, Ser54, Thr54, Gln54, Phe54, Met54, or Leu54 and/or an Arg at position 56 (Arg 56); and (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 90. In particular embodiments, the VH comprises an amino acid sequence selected from SEQ ID NO 87, SEQ ID NO 88 and SEQ ID NO 89. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO 90. In a particular embodiment, the antibody comprises the VH sequence of SEQ ID NO. 89 and the VL sequence of SEQ ID NO. 90.

In particular embodiments, the antibodies of the invention specifically bind to HCMV complex I on the surface of HCMV and neutralize HCMV with an EC90 of 0.1 μ g/ml or less. In particular embodiments, the isolated anti-complex I antibodies of the invention specifically bind to HCMV complex I on the surface of the HCMV and are present at an antibody concentration of 0.05 μ g/ml, 0.02 μ g/ml, 0.015 μ g/ml, 0.014 μ g/ml, 0.013 μ g/ml, 0.012 μ g/ml, 0.011 μ g/ml, 0.010 μ g/ml, 0.009 μ g/ml, 0.008 μ g/ml, 0.007 μ g/ml, 0.006 μ g/ml, 0.005 μ g/ml, 0.004 μ g/ml, 0.003 μ g/ml, 0.002 μ g/ml, 0.001 μ g/ml, 0.0009 μ g/ml, 0.0008 μ g/ml, 0.0007 μ g/ml or less (e.g., at an antibody concentration of 10 μ g/ml, 0.015 μ g/ml, 0.014 μ g/ml, 0.006 μ g/ml, 0.01 μ g/ml, or less)^-8M、10^-9M10^-10M、10^-11M、10^-12M、10^-13M or lower antibody concentration) and 50% HCMV.

In particular embodiments, the isolated anti-gH antibodies of the invention specifically bind HCMV gH. The antibody binds to gH on the surface of HCMV and neutralizes HCMV with an EC90 of 1 μ g/ml or less. The isolated anti-gH antibodies of the invention bind to gH on the surface of HCMVAnd at an antibody concentration of 0.1. mu.g/ml, 0.09. mu.g/ml, 0.08. mu.g/ml, 0.07. mu.g/ml, 0.06. mu.g/ml, 0.05. mu.g/ml, 0.04. mu.g/ml, 0.03. mu.g/ml, 0.02. mu.g/ml, 0.015. mu.g/ml, 0.014. mu.g/ml, 0.013. mu.g/ml, 0.012. mu.g/ml, 0.011. mu.g/ml, 0.010. mu.g/ml, 0.009. mu.g/ml, 0.008. mu.g/ml, 0.007. mu.g/ml, 0.006. mu.g/ml, 0.005. mu.g/ml, 0.004. mu.g/ml, 0.003. mu.g/ml, 0.002. mu.g/ml, 0.001. mu.g/ml or less (e.g/ml, 10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M or lower antibody concentration) and 50% HCMV.

The antibodies of the invention may be monoclonal antibodies, including, for example, human, humanized or chimeric antibodies. The invention also provides antibody fragments that specifically bind HCMV gH and/or complex I.

In particular embodiments, the antibody that specifically binds HCMV complex I and/or gH is a full-length IgG1 antibody.

The invention also provides isolated nucleic acids encoding antibodies that specifically bind to HCMV complex I and/or gH. The invention also provides host cells comprising nucleic acids encoding such antibodies.

The invention further provides a method of producing an antibody comprising culturing a host cell comprising a nucleic acid encoding an antibody that specifically binds complex I and/or gH, thereby allowing production of the antibody. The method may further comprise recovering the antibody from the host cell.

The invention also provides pharmaceutical formulations comprising a combination of an anti-complex I antibody or an anti-gH antibody or an anti-complex I antibody and an anti-gH antibody and a pharmaceutically acceptable carrier. The pharmaceutical formulations of the respective antibodies may be separate or combined. The pharmaceutical formulation may further comprise additional therapeutic agents (e.g., ganciclovir (ganciclovir), foscarnet (foscarnet), valganciclovir (valganciclovir) and cidofovir (cidofovir)).

The invention also provides compositions comprising anti-complex I antibodies, or anti-gH antibodies, or a combination of anti-complex I antibodies and anti-gH antibodies. The compositions comprising the respective antibodies may be separate or combined. The composition may further comprise an additional therapeutic agent (e.g., ganciclovir, foscarnetil, valganciclovir, and silurovir).

The invention also provides compositions comprising anti-complex I and/or anti-gH antibodies for use in inhibiting, treating, or preventing HCMV infection. In some embodiments, the use is for inhibiting, treating, or preventing congenital HCMV infection, or for inhibiting, treating, or preventing HCMV infection in a transplanted tissue, organ, or donor infected with HCMV or a tissue or organ transplant recipient that has been infected with HCMV. Additional embodiments include uses wherein the transplant recipient has been previously infected with HCMV and is at risk of reactivation. In particular embodiments, the tissue or organ transplant recipient is seronegative for HCMV infection. In particular embodiments, the composition comprising an antibody that binds HCMVgH is separate from the composition comprising an antibody that binds HCMV complex I.

Compositions comprising the antibodies of the invention may also be used in the manufacture of a medicament. The medicament may be for use in the treatment, inhibition or prevention of HCMV infection, for example to inhibit, prevent or treat congenital HCMV infection, or HCMV infection in an organ or tissue transplant recipient for which the transplanted organ, tissue or donor is or has been infected with HCMV. In another embodiment, the transplant recipient has been previously infected with HCMV and is at risk of reactivation. In particular embodiments, the medicament may further comprise an additional therapeutic agent (e.g., ganciclovir, foscarnetil, valganciclovir, and sildenafil). In particular embodiments, the tissue or organ transplant recipient is seronegative for HCMV infection. In particular embodiments, the composition comprising an antibody that binds HCMVgH is in a separate composition from the antibody that binds HCMV complex I.

The invention also provides a method of treating, inhibiting, or preventing HCMV infection comprising administering to a patient an effective amount of a composition comprising an anti-gH antibody, an anti-complex I antibody, or a combination thereof. The invention also provides a method of treating, inhibiting, or preventing congenital HCMV infection, comprising administering to a pregnant woman an effective amount of a composition comprising an antibody of the invention, or a combination thereof. The invention also provides a method of treating a HCMV-infected fetus comprising administering to a pregnant woman an effective amount of a composition comprising an antibody of the invention or a combination thereof. The invention also provides a method of treating an infant infected with HCMV or an infant that has been exposed to HCMV during gestation comprising administering to the infant an effective amount of a composition comprising an antibody of the invention or a combination thereof.

The invention also provides a method of treating, inhibiting or preventing HCMV infection in an organ or tissue transplant recipient comprising administering to the recipient having the organ or tissue transplanted therewith a composition comprising an antibody of the invention or a combination thereof in an amount effective to treat, inhibit or prevent HCMV infection caused by an organ or tissue obtained from an organ donor or tissue donor infected with HCMV or already infected with HCMV. Additional embodiments include methods wherein the transplant recipient has previously been infected with HCMV and is at risk of reactivation. The method of treatment may further comprise administering to the patient an additional therapeutic agent (e.g., ganciclovir, foscarnetil, valganciclovir, and silurovir).

In particular embodiments, the composition comprising an antibody that binds HCMV gH is in a separate composition from the composition comprising an antibody that binds HCMV complex I. In other embodiments, the composition comprising an antibody that binds HCMV gH is administered simultaneously with, before, or after the administration of the composition comprising an antibody that binds HCMV complex I.

The invention also provides anti-complex I and/or anti-gH antibodies for use in inhibiting, treating or preventing HCMV infection. In some embodiments, the use is for inhibiting, treating, or preventing a congenital HCMV infection, or an HCMV infection in a tissue or organ transplant recipient for which the transplanted tissue, organ or donor is or has been infected with HCMV. Additional embodiments include uses wherein the transplant recipient has been previously infected with HCMV and is at risk of reactivation. In particular embodiments, the tissue or organ transplant recipient is seronegative for HCMV infection.

The antibodies of the invention may also be used in the manufacture of a medicament. The medicament may be for use in the treatment, inhibition or prevention of HCMV infection, for example to inhibit, prevent or treat congenital HCMV infection or HCMV infection in an organ or tissue transplant recipient for which the transplanted organ, tissue or donor is or has been infected with HCMV. In another embodiment, the transplant recipient has been previously infected with HCMV and is at risk of reactivation. In particular embodiments, the medicament may further comprise an additional therapeutic agent (e.g., ganciclovir, foscarnetil, valganciclovir, and sildenafil). In particular embodiments, the tissue or organ transplant recipient is seronegative for HCMV infection.

The invention also provides a method of treating, inhibiting, or preventing HCMV infection comprising administering to a patient an effective amount of an anti-gH antibody, an anti-complex I antibody, or a combination thereof. The invention also provides a method of treating, inhibiting, or preventing congenital HCMV infection, comprising administering to a pregnant woman an effective amount of an antibody of the invention, or a combination thereof. The invention also provides a method of treating a HCMV-infected fetus comprising administering to a pregnant woman an effective amount of an antibody of the invention, or a combination thereof.

The invention also provides a method of treating, inhibiting or preventing HCMV infection in an organ or tissue transplant recipient comprising administering to the organ or tissue transplant recipient an effective amount of an antibody of the invention, or a combination thereof, to treat, inhibit or prevent HCMV infection caused by an organ or tissue from an organ donor or tissue donor infected or already infected with HCMV. Additional embodiments include methods wherein the transplant recipient has previously been infected with HCMV and is at risk of reactivation. The method of treatment may further comprise administering to the patient an additional therapeutic agent (e.g., ganciclovir, foscarnetil, valganciclovir, and silurovir).

In particular embodiments, the antibody that binds HCMV gH is administered separately from the antibody that binds HCMV complex I. In other embodiments, the antibody that binds HCMV gH is administered simultaneously, prior to, or after the antibody that binds HCMV complex I.

In particular embodiments, the organ transplant is a heart, kidney, liver, lung, pancreas, intestine, or thymus. In other embodiments, the tissue graft is a hand, cornea, skin, face, pancreatic islets, bone marrow, stem cells, whole blood, platelets, serum, blood cells, blood vessels, heart valves, bone progenitor cells, cartilage, ligament, tendon, muscle lining (muscle lining).

The invention also provides antibodies that bind to the same epitope as the anti-gH and/or anti-complex I antibodies of the invention. Additional embodiments include antibodies that bind to an HCMV gH epitope comprising amino acids corresponding to amino acids selected from the group consisting of: tryptophan at position 168 of SEQ ID NO. 1; 1, aspartic acid at position 446 of SEQ ID NO; (ii) proline at position 171 of SEQ ID NO: 1; and combinations thereof. Additional embodiments include antibodies that bind to an epitope of HCMV complex I comprising amino acids corresponding to amino acids selected from the group consisting of: a glutamine at position 47 of SEQ ID NO. 203; (ii) a lysine at position 51 of SEQ ID NO. 203; (iii) (ii) aspartic acid at position 46 of SEQ ID No. 203; and (iv) combinations thereof. Additional embodiments include antibodies that bind to a polypeptide of HCMV complex I, wherein the polypeptide comprises amino acid sequence SRALPDQTRYKYVEQLVDLTLNYHYDAS (SEQ ID NO: 194).

Brief Description of Drawings

FIG. 1 shows the heavy chain variable region (VH) of murine mAb8G8 (SEQ ID NO: 50) in combination with a selected human heavy chain variable region: alignment of the amino acid sequences of VH1FW (SEQ ID NO: 52), human VH3FW (SEQ ID NO: 53), and human VH7FW (SEQ ID NO: 54). Amino acids are numbered according to Kabat numbering. Hypervariable regions (HVRs) are boxed. Circles indicate VL-VH interactions (Padlan (1994) mol. Immunol.31: 169); the two asterisks (one on top of the other) indicate the Vernier position (Foote and Winter (1992) J.mol.biol.224: 487) and the FW-CDR interaction (Padlan (1994) mol.Immunol.31: 169). A single asterisk at positions 47, 64, 66, 68 indicates the Vernier position (Foote and Winter (1992) J.Mol.biol.224: 487); a single asterisk at position 58 indicates FW-CDR interaction (Padlan (1994) mol. Immunol.31: 169).

FIG. 2 shows an alignment of the amino acid sequences of the light chain variable region (VL) of murine mAb8G8 (SEQ ID NO: 51) with the human light chain variable region λ 3FW region (SEQ ID NO: 69) and the human λ 4FW region (SEQ ID NO: 55). Amino acids are numbered according to Kabat numbering. Hypervariable regions (HVRs) are boxed. Circles indicate VL-VH interactions (Padlan (1994) mol. Immunol.31: 169); the two asterisks (one on top of the other) indicate the Vernier position (Foote and Winter (1992) J.mol.biol.224: 487) and the FW-CDR interaction (Padlan (1994) mol.Immunol.31: 169). A single asterisk at positions 47, 64, 66, 68 indicates the Vernier position (Foote and Winter (1992) J.Mol.biol.224: 487); a single asterisk at position 58 indicates FW-CDR interaction (Padlan (1994) mol. Immunol.31: 169).

Figure 3 shows the results of the neutralization assay test comparing the 8G8 λ 3 variant with the 8G8 λ 4 variant. Small graph A: in addition to the mouse/human chimeric 8G8 antibody (QE 7/C2), a humanized 8G8 λ 3 antibody with human VH1, VH3 or VH7 was also used in the neutralization assay. Small graph B: in addition to the mouse/human chimeric 8G8 antibody (QE 7/C2), humanized 8G8 λ 4 antibodies with human VH1, VH3 or VH7 were also used in neutralization assay assays. EC50 values for the experiments are shown below the corresponding experiments.

FIG. 4 shows the sequence of a mutant of 8G8 HVR-L2. The amino acid sequence of HVR-L2 and the first amino acid of FR3 are shown (WT, SEQ ID NO: 57; A1, SEQ ID NO: 58; E1, SEQ ID NO: 59; T1, SEQ ID NO: 60; A2, SEQ ID NO: 61; E2, SEQ ID NO: 62; T2, SEQ ID NO: 63; SG, SEQ ID NO: 64; SGSG, SEQ ID NO: 65; TGDA, SEQ ID NO: 66). The numbering in this figure is based on Kabat numbering.

Figure 5 shows the results of a neutralization assay using a variety of humanized 8G8 antibodies having the mutated HVR-L2 region shown in figure 4 containing a single amino acid substitution. Small graph A: neutralization assay test. The HVR-L2 mutant antibodies all contained human 8G8VH1 chain. Small graph B: EC50 values for the experiment.

Figure 6 shows the results of a neutralization assay using a variety of humanized 8G8 antibodies having the mutated HVR-L2 region shown in figure 4 containing two amino acid substitutions. Small graph A: neutralization assay test. The HVR-L2 mutant antibodies all contained human 8G8VH1 chain. Small graph B: EC50 values for the experiment.

FIG. 7 shows an amino acid sequence alignment of the light chain variable region of murine mAb8G8 (SEQ ID NO: 51) with the human light chain variable region, λ 4FW (SEQ ID NO: 55) and the humanized light chain variable region of 8G8 (hu8G8. λ 4 FW) (SEQ ID NO: 48) at λ 4 FW. Amino acids are numbered according to Kabat numbering. Hypervariable regions (HVRs) are boxed. Circles indicate VL-VH interactions (Padlan (1994) mol. Immunol.31: 169); the two asterisks (one on top of the other) indicate the Vernier position (Foote and Winter (1992) J.mol.biol.224: 487) and the FW-CDR interaction (Padlan (1994) mol.Immunol.31: 169). A single asterisk at positions 47, 64, 66, 68 indicates the Vernier position (Foote and Winter (1992) J.Mol.biol.224: 487); a single asterisk at position 58 indicates FW-CDR interaction (Padlan (1994) mol. Immunol.31: 169).

FIG. 8 shows an alignment of the amino acid sequences of the heavy chain variable region of murine mAb8G8 (SEQ ID NO: 50) with the human heavy chain variable region VH1 framework (VH 1 FW) (SEQ ID NO: 52) and the humanized heavy chain variable region of 8G8 on VH1FW (hu8G8. VH1) (SEQ ID NO: 45). Amino acids are numbered according to Kabat numbering. Hypervariable regions (HVRs) are boxed. Circles indicate VL-VH interactions (Padlan (1994) mol. Immunol.31: 169); the two asterisks (one on top of the other) indicate the Vernier position (Foote and Winter (1992) J.mol.biol.224: 487) and the FW-CDR interaction (Padlan (1994) mol.Immunol.31: 169). A single asterisk at positions 47, 64, 66, 68 indicates the Vernier position (Foote and Winter (1992) J.Mol.biol.224: 487); a single asterisk at position 58 indicates FW-CDR interaction (Padlan (1994) mol. Immunol.31: 169). An exemplary nucleic acid sequence encoding hu8G8.VH1 is also shown (SEQ ID NO: 185).

FIG. 9 shows an amino acid sequence alignment of the heavy chain variable region of murine mAb8G8 (SEQ ID NO: 50) with human heavy chain variable region VH3FW (SEQ ID NO: 53) and the humanized heavy chain variable region of 8G8 on VH3FW (hu8G8. VH3) (SEQ ID NO: 46). Amino acids are numbered according to Kabat numbering. Hypervariable regions (HVRs) are boxed. Circles indicate VL-VH interactions (Padlan (1994) mol. Immunol.31: 169); the two asterisks (one on top of the other) indicate the Vernier position (Foote and Winter (1992) J.mol.biol.224: 487) and the FW-CDR interaction (Padlan (1994) mol.Immunol.31: 169). A single asterisk at positions 47, 64, 66, 68 indicates the Vernier position (Foote and Winter (1992) J.Mol.biol.224: 487); a single asterisk at position 58 indicates FW-CDR interaction (Padlan (1994) mol. Immunol.31: 169).

FIG. 10 shows an amino acid sequence alignment of the light chain variable region of murine mAb8G8VL (SEQ ID NO: 51) with the light chain variable region of the λ 4FW region (SEQ ID NO: 55) and the humanized light chain variable region of 8G8 (λ 48G8 graft) (SEQ ID NO: 49) on λ 4FW, in which amino acid changes were introduced at amino acids 2 and 36 according to Kabat numbering. Amino acids are numbered according to Kabat numbering. Hypervariable regions (HVRs) are boxed. Circles indicate VL-VH interactions (Padlan (1994) mol. Immunol.31: 169); the two asterisks (one on top of the other) indicate the Vernier position (Foote and Winter (1992) J.mol.biol.224: 487) and the FW-CDR interaction (Padlan (1994) mol.Immunol.31: 169). A single asterisk at positions 47, 64, 66, 68 indicates the Vernier position (Foote and Winter (1992) J.Mol.biol.224: 487); a single asterisk at position 58 indicates FW-CDR interaction (Padlan (1994) mol. Immunol.31: 169). An exemplary nucleic acid sequence encoding the lambda 48G8 graft is also shown (SEQ ID NO: 186).

FIG. 11 shows an alignment of the amino acid sequences of human antibody MSL-109 and mAb HB 1. Small graph A: alignment of MSL-109VL (SEQ ID NO: 90) with affinity matured HB1VL (also SEQ ID NO:90 (100% identity)); small graph B: the human antibody MSL-109VH (SEQ ID NO: 92) was aligned with the amino acid sequence of affinity matured HB1VH (SEQ ID NO: 89). Amino acids are numbered according to Kabat numbering. Hypervariable regions (HVRs) are boxed.

FIG. 12A shows the amino acid sequence of HVR-H2 from MSL-109 (SEQ ID NO: 91) and IGHV3-21 x 01 (SEQ ID NO: 93) and the various amino acid substitutions made. Fig. 12B and 12C show the results of two different neutralization assay experiments performed using antibodies containing mutated HVR-H2 regions. Neutralization assay assays with Fab (fig. 12B) and mAb (fig. 12C) are shown. IC50 is provided in nM units.

Fig. 13 shows the results of neutralization assay comparing antibodies containing λ 48G8 graft and hu8g8.vh1 (hereinafter "hu 8G 8") and HB1 to HIG for their ability to prevent infection of epithelial cells and fibroblasts.

Figure 14 shows the results of a virus neutralization assay using depleted hyperimmune globulin (HIG) on epithelial and fibroblasts. HIG-depleted anti-gB-specific antibodies, anti-complex I-specific antibodies, anti-gH/gL antibodies, or as controls for simulated depletion.

Fig. 15 shows FACS analysis results, compared to known anti-gB, anti-gH and anti-UL 131 antibodies, to determine the antigen specificity of HB1 and hu8G8 antibodies. APC intensity on the x-axis indicates binding of the antibody. The y-axis indicates the proportion of cells at a given intensity, expressed as a percentage of the maximum number of cells at any intensity.

FIG. 16 shows the results of a neutralization assay in which hu8G8 and HB1 were mixed at a 1:1 ratio and tested in a dilution series for their ability to inhibit HCMV infection of epithelial cells. The combination of the two antibodies had an additive effect and it had a behaviour according to the Bliss independence equation (Bliss independence response) (C = a + B-a × B for the combined response C of the two individual compounds with effects a and B).

Fig. 17 shows the results of a neutralization assay test to determine HB1 potency in combination with various concentrations of hu8G8, or hu8G8 potency in combination with various concentrations of HB 1.

FIG. 18 shows the results of a neutralization assay test with HB1 resistant HCMV mutants. Panel a shows the results of a neutralization assay using HB1 antibody. Panel B shows the results of the neutralization assay using hu8G8 antibody. The HB1 resistant HCMV mutant was still sensitive to neutralization by hu8G8.

FIG. 19 shows the results of a neutralization assay test with hu8G8 resistant HCMV mutants. Panel a shows the results of a neutralization assay using HB1 antibody. Panel B shows the results of the neutralization assay using hu8G8 antibody. The hu8G8 resistant HCMV mutant was still sensitive to neutralization by HB 1.

FIG. 20 shows data on viral entry of HCMV strain (WT) D1 (VR 1814 grown in parallel when producing resistant strain) compared to multiple HB1 resistant viral mutants on epithelial and fibroblast cells.

FIG. 21 shows the ability of the HB1 antibody to bind cell surface-expressed gH/gL containing a resistance-conferring point mutation in gH as determined by FACS analysis. Different anti-gH antibodies were used as positive controls for cell surface expression. The x-axis is GFP intensity, indicating expression of HCMV glycoprotein. The y-axis is the APC signal indicating antibody binding.

FIG. 22 shows the ability of the hu8G8 antibody to bind to cell surface expressed complex I containing a point mutation in complex I conferring resistance as determined by FACS analysis. anti-UL 131 antibody and anti-gH antibody were used as positive controls for cell surface expression. The x-axis is the GFP intensity indicating HCMV glycoprotein expression. The y-axis is the APC signal indicating antibody binding.

Fig. 23A and B show the results of Scatchard analysis to determine the binding affinity of hu8G8 and HB1 for their antigens. The results were plotted using the fitting algorithm of Munson and Rodbard. With combined y-axis indication¹²⁵I concentration ratio of labeled antibody to total antibody. The total antibody is calculated as¹²⁵I markingAnd concentration of unlabeled antibody.

FIG. 24 shows the results of an ELISA assay measuring the binding of hu8G8 and a positive control antibody (anti-HIS) to a peptide fragment of UL131 (amino acid 41 (Ser) to amino acid 68 (Ser) of SEQ ID NO: 194) (SRA-Helix WT) or the corresponding fragment containing the amino acid substitution Q47K (SRA-Helix Mut).

Detailed description of embodiments of the invention

I. Definition of

An "acceptor human framework" for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework (as defined below). An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence as it, or it may contain amino acid sequence variations. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

"affinity" refers to the sum of the forces of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

An "affinity matured" antibody refers to: an antibody having one or more alterations in one or more hypervariable regions (HVRs) which result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody not having such alterations.

The terms "anti-complex I antibody" and "antibody that binds to complex I" refer to an antibody that is capable of binding to complex I with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent targeting complex I. In one embodiment, the extent of binding of the anti-complex I antibody to an unrelated non-complex I protein is less than about 10% of the binding of the antibody to complex I as measured by, for example, Radioimmunoassay (RIA). In particular embodiments, the antibody that binds to Complex I has ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (Kd). In particular embodiments, the anti-complex I antibody binds to a complex I epitope that is conserved among human CMV isolates. In particular embodiments, the anti-complex I antibody binds to a complex I epitope that is conserved among CMV strains infecting different species. In particular embodiments, an "anti-complex I antibody" binds a conformational epitope of complex I, and in particular embodiments, an anti-complex I antibody binds an epitope within a single complex I protein member (i.e., gL, UL128, UL130, or UL 131) that is not gH.

The terms "anti-gH antibody" and "antibody that binds to gH" refer to an antibody that is capable of binding gH with sufficient affinity that the antibody can be used as a diagnostic and/or therapeutic agent that targets gH. In one embodiment, the extent of binding of the anti-gH antibody to an unrelated, non-gH protein is less than about 10% of the binding of the antibody to gH, as measured, for example, by Radioimmunoassay (RIA). In particular embodiments, an antibody that binds to gH has ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13Solution of M)The dissociation constant (Kd). In particular embodiments, the anti-gH antibody binds to a gH epitope conserved among human CMV isolates. In particular embodiments, the anti-gH antibody binds to a gH epitope that is conserved among CMV strains infecting different species.

The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

An "antibody fragment" refers to a molecule that is not an intact antibody, which comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab')₂(ii) a Diabodies (diabodies); a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks 50% or more of the binding of the reference antibody to its antigen in a competition assay, and conversely, the reference antibody blocks 50% or more of the binding of the antibody to its antigen in a competition assay. Exemplary competition assay assays are provided herein.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain has. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The heavy chain constant domains corresponding to different classes of immunoglobulins are designated α, γ, and μ, respectively.

As used herein, the term "complex I", unless otherwise specified, refers to any native complex I from any cytomegalovirus source, including CMV that infects mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses the combination of all of the gH, gL, UL128, UL130 and UL131 polypeptides. The term also includes naturally occurring variants of the protein of complex I, such as splice variants or allelic variants. The amino acid sequence of an exemplary HCMV gH is shown in SEQ ID NO 1. The amino acid sequence of an exemplary HCMV gL is shown in SEQ ID NO: 2. The amino acid sequence of an exemplary HCMV UL128 is shown in SEQ ID NO 3. The amino acid sequence of an exemplary HCMV UL130 is shown in SEQ ID NO 4. The amino acid sequence of an exemplary HCMV UL131 is shown in SEQ ID NO 5. Additional exemplary sequences for HCMV gH, gL, UL128, UL130 and UL131 may be found in Genbank accession number GU179289 (Dargan et al, J.Gen.Virol.91: 1535-1546 (2010)), which are all incorporated herein by reference in their entirety and as SEQ ID NO: 206 (gH), seq id NO: 208 (gL), SEQ ID NO: 205 (UL 128), SEQ ID NO: 204 (UL 130); and SEQ ID NO:203 (UL 131) is included herein.

As used herein, unless otherwise specified, the term "complex II" refers to any native complex II from any cytomegalovirus source, including CMV that infects mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses all combinations of gH, gL and gO. The term also includes naturally occurring variants of the protein of complex II, such as splice variants or allelic variants. The amino acid sequence of an exemplary HCMV gH is shown in SEQ ID NO 1. The amino acid sequence of an exemplary HCMV gL is shown in SEQ ID NO 2. The amino acid sequence of an exemplary HCMV gO is shown in SEQ ID NO: 209. Additional exemplary sequences for HCMV gH, gL and gO may be found in Genbank accession number GU179289 (Dargan et al, J.Gen.Virol.91: 1535-1546 (2010)), which are all incorporated herein by reference in their entirety and as SEQ ID NO: 206 (gH), SEQ ID NO: 208 (gL) and seq id NO: 207 (gO) is included herein.

As used herein, unless otherwise specified, the term "gH" refers to any native gH from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" unprocessed gH as well as any gH form that results from processing in the cell. The term also encompasses naturally occurring gH variants, such as splice variants or allelic variants. The amino acid sequence of gH is about 95% identical between CMV isolates. The amino acid sequence of an exemplary HCMV gH is shown in SEQ ID NO 1. Additional exemplary sequences for HCMV gH may be found in Genbank accession number GU179289 (Dargan et al, J.Gen.Virol.91: 1535-1546 (2010)), which are all incorporated herein by reference in their entirety and as SEQ ID NO: 206 (gH) is included herein.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At)²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin (adriamicin), vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin (doxorubicin), melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); a growth inhibitor; enzymes and fragments thereof such as nucleolytic enzymes; (ii) an antibiotic; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various anti-tumor or anti-cancer agents disclosed below.

"effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

An "effective amount" of an active agent, e.g., a pharmaceutical formulation, is an amount effective to achieve the desired therapeutic or prophylactic result at the desired dosage and for the desired period of time.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which contains at least part of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys 447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, published Health Service 5 th edition, National Institutes of Health, Bethesda, MD, 1991.

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain generally consist of four FR domains: FR1, FR2, FR3 and FR 4. Accordingly, HVR and FR sequences typically occur in VH (or VL) in the following order: FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR 4.

The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as that screened or selected for in the originally transformed cell are included herein.

A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using human antibody repertoires (antibodies polypeptides) or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues.

A "human consensus framework" is a framework representing the amino acid residues most commonly occurring in the framework sequences of a selected human immunoglobulin VL or VH. Typically, the selected human immunoglobulin VL or VH sequence is from a subgroup of variable domain sequences. Typically, this sequence subset is a subset as in Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, NIH Publication91-3242, Bethesda MD (1991), volumes 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al, supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al, supra.

"humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In particular embodiments, the humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies, e.g., non-human antibodies, refer to antibodies that have undergone humanization.

As used herein, the term "hypervariable region" or "HVR" refers to the various regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Generally, a native four-chain antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically contain amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs) which have the highest sequence variability and/or are involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3). (Chothia and Lesk, J.mol.biol.196:901-917 (1987).) exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 50-65 of 31-35B, H2 of H1 and 95-102 of H3. (Kabat et al, Sequences of Proteins of Immunological Interest, published Health Service 5 th edition, National Institutes of Health, Bethesda, Md. (1991)) in addition to the CDR1 in the VH, the CDRs generally contain amino acid residues that form a hypervariable loop. The CDRs also contain "specificity determining residues" or "SDRs," which are residues that contact the antigen. SDRs are contained within regions of CDRs called abbreviated CDRs (abbrevated-CDRs) or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2 and a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 50-58 of 31-35B, H2 of H1 and 95-102 of H3. (see Almagro and Fransson, front. biosci.13:1619-1633 (2008)), unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al, supra.

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules (including but not limited to cytotoxic agents).

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular embodiments, the individual or subject is a human.

As used herein, "infant" refers to an individual or subject ranging in age from birth to no more than about one year of age, including infants from 0 to about 12 months.

An "isolated" antibody is one that has been separated from components in its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, j.chromanogr.b 848:79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

"isolated nucleic acid encoding an anti-complex I antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

"isolated nucleic acid encoding an anti-gH antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., each individual antibody comprised in the population is identical and/or binds to the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or occurring during the production of a monoclonal antibody preparation, and such variants are typically present in minute amounts. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

"naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (motif), such as a cytotoxic moiety, or to a radiolabel. Naked antibodies may be present in pharmaceutical formulations.

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, native IgG antibodies are heterotetrameric proteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-linked. From N to C-terminus, each heavy chain has a variable region (VH), also known as the variable heavy or variable domain, followed by three constant domains (CH 1, CH2, and CH 3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a constant light Chain (CL) domain. The light chain of an antibody can be assigned to one of two types called kappa (κ) and lambda (λ) based on the amino acid sequence of its constant domain.

The term "package insert" is used to refer to an insert customarily included in commercial packages of therapeutic products, that contains information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as: after aligning the candidate and reference sequences and introducing gaps, if necessary, to achieve a maximum percent sequence identity, no conservative substitutions are considered as part of the sequence identity, the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence. Alignments for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences to be compared. However, for purposes herein,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, inc, and the source code has been submitted to the us copyright Office (u.s.copy Office, washington d.c., 20559) along with a user manual, registered under us copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including the digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of ALIGN-2 for amino acid sequence comparison, the% amino acid sequence identity of a given amino acid sequence A with, or relative to a given amino acid sequence B (which may alternatively be expressed as: a given amino acid sequence A with, or with or comprising a particular% amino acid sequence identity relative to a given amino acid sequence B) is calculated as follows:

100 times a fraction X/Y

Wherein X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the ALIGN-2 program alignment of a and B, and wherein Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A relative to B will not be equal to the% amino acid sequence identity of B relative to A. Unless specifically stated otherwise, all% amino acid sequence identity values used herein were obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

The term "pharmaceutical formulation" refers to a preparation having a form that allows the biological activity of the active ingredient contained in the preparation to be effective, and which does not contain additional components having unacceptable toxicity to the subject to which the formulation is to be administered.

"pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is not toxic to the subject, except for the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, "treatment" (and grammatical variants thereof, such as "treating" or "treating") refers to clinical intervention in which the force profile alters the natural processes of the treated individual, which may be performed for prophylaxis or during clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating a disease state, and remission or improved prognosis. In some embodiments, the compositions of the invention are used to delay the progression of the disease or slow the progression of the disease or reduce the incidence of the disease or severity of the disease symptoms.

The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved Framework Regions (FRs) and three hypervariable regions (HVRs). (see, e.g., Kindt et al Kuby Immunology, 6th edition, W.H.Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to an antigen can be isolated by screening a library of complementary VH or VL domains, respectively, using VH or VL domains from antibodies that bind the particular antigen. See, e.g., Portolano et al, J.Immunol.150: 880-; clarkson et al, Nature352: 624-.

As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors which are autonomously replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

Compositions and methods

In one aspect, the invention is based, in part, on the discovery of monoclonal antibodies that neutralize HCMV infection. In particular embodiments, antibodies that bind to complex I are provided. In other embodiments, antibodies that bind gH are provided. The antibodies of the invention may be used, for example, to prevent, inhibit and/or treat HCMV infection, congenital HCMV infection and infection of a patient by HCMV-infected transplanted tissue. Antibodies may also be used to diagnose HCMV infection.

In one aspect, the invention is further based, in part, on the discovery of compositions comprising a combination of monoclonal antibodies that can inhibit HCMV virus entry into all cell types of the placenta: endothelial cells, epithelial cells, monocytes/macrophages and fibroblasts, and reduces and/or inhibits the formation of HCMV resistant strains. In particular embodiments, methods of using these compositions are provided. The compositions of the invention may, for example, be used to prevent, inhibit and/or treat HCMV infection, congenital HCMV infection and infection in a patient by HCMV-infected transplanted organs or tissues harvested from a patient previously or now infected with HCMV. The compositions may also be used to diagnose HCMV infection.

A. Exemplary anti-Complex I antibodies

In one aspect, the invention provides an isolated antibody that binds to complex I. In particular embodiments, antiThe complex I antibody specifically binds to a conformational epitope formed by the association of UL128, UL130, UL131 and gH/gL, or to an epitope within the individual members of complex I. In some embodiments, the anti-complex I antibody neutralizes HCMV with an EC90 of 0.7. mu.g/ml, 0.5. mu.g/ml, 0.3. mu.g/ml, 0.1. mu.g/ml, 0.09. mu.g/ml, 0.08. mu.g/ml, 0.07. mu.g/ml, 0.06. mu.g/ml, 0.05. mu.g/ml, 0.04. mu.g/ml, 0.03. mu.g/ml, 0.02. mu.g/ml, 0.015, 0.012. mu.g/ml, 0.011. mu.g/ml, 0.010. mu.g/ml or less. In other aspects, the anti-complex I antibody specifically binds to complex I on the surface of HCMV and at an antibody concentration of 0.05 μ g/ml, 0.02 μ g/ml, 0.015 μ g/ml, 0.014 μ g/ml, 0.013 μ g/ml, 0.012 μ g/ml, 0.011 μ g/ml, 0.010 μ g/ml, 0.009 μ g/ml, 0.008 μ g/ml, 0.007 μ g/ml, 0.006 μ g/ml, 0.005 μ g/ml, 0.004 μ g/ml, 0.003 μ g/ml, 0.002 μ g/ml, 0.001 μ g/ml, 0.0009 μ g/ml, 0.0008 μ g/ml, 0.0007 μ g/ml or less (e.g., at 10 μ g/ml, e.g., at^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M or lower antibody concentration), and 50% HCMV was neutralized.

In one aspect, the invention provides anti-complex I antibodies comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 8; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 9; (e) HVR-L2 comprising an amino acid sequence selected from SEQ ID NOs: 10-19; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8.

In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 9; (b) HVR-L2 comprising an amino acid sequence selected from SEQ ID NOs: 10-19; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In one embodiment, the antibody comprises all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:8, and three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In another embodiment, the antibody comprises all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:8, and three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 11; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In another embodiment, the antibody comprises all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:8, and three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO 12; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In another embodiment, the antibody comprises all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:8, and three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO 13; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In another embodiment, the antibody comprises all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:8, and three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 14; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In another embodiment, the antibody comprises all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:8, and three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 15; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In another embodiment, the antibody comprises all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:8, and three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO 16; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In another embodiment, the antibody comprises all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:8, and three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO 17; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In another embodiment, the antibody comprises all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:8, and three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 18; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In another embodiment, the antibody comprises all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:8, and three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 19; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In some embodiments, the antibody comprises all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:8, and three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO 21 and the first amino acid of the light chain variable region framework FR 3; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20. In particular embodiments, any one or more amino acids of an anti-complex I antibody as provided above are substituted at the following HVR positions: in HVR-L2 (SEQ ID NO: 10): positions 4,5, 11 and 12. In particular embodiments, the substitutions are conservative substitutions, as provided herein. In particular embodiments, any one or more of the following substitutions may be made in any combination: in HVR-L2 (SEQ ID NO: 57): D4E, D4T, D4S, G5A, D11E, D11T, D11S, and G12A. The consensus sequence of SEQ ID NO 21 covers all possible combinations of the above substitutions.

In any of the embodiments above, the anti-complex I antibody is humanized. In one embodiment, the anti-complex I antibody comprises HVRs as in any of the embodiments above, and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-Complex I antibody comprises the HVRs as in any of the embodiments above, and further comprises a VH comprising the FR1 sequence of SEQ ID NO:22, the FR2 sequence of SEQ ID NO:23, the FR3 sequence of SEQ ID NO:24, and the FR4 sequence of SEQ ID NO: 25. In other embodiments, the anti-complex I antibody comprises the HVRs of any of the embodiments described above, and further comprises a VH comprising the FR1 sequence of SEQ ID NO:22, the FR2 sequence of SEQ ID NO:27, the FR3 sequence of SEQ ID NO:28, and the FR4 sequence of SEQ ID NO: 29. In other embodiments, the anti-complex I antibody comprises the HVRs of any of the embodiments described above, and further comprises a VH comprising the FR1 sequence of SEQ ID NO:30, the FR2 sequence of SEQ ID NO:31, the FR3 sequence of SEQ ID NO:32, and the FR4 sequence of SEQ ID NO: 25. In other embodiments, the anti-complex I antibody comprises the HVRs of any of the embodiments described above, and further comprises a VH comprising the FR1 sequence of SEQ ID NO:33, the FR2 sequence of SEQ ID NO:23, the FR3 sequence of SEQ ID NO:34, and the FR4 sequence of SEQ ID NO: 25.

In another embodiment, the anti-Complex I antibody comprises the HVRs of any of the embodiments described above, and further comprises a VL comprising the FR1 sequence of SEQ ID NO:35, the FR2 sequence of SEQ ID NO:36, the FR3 sequence of SEQ ID NO:37, and the FR4 sequence of SEQ ID NO: 38. In other embodiments, the anti-complex I antibody comprises the HVRs of any of the embodiments described above, and further comprises a VL comprising the FR1 sequence of SEQ ID NO:39, the FR2 sequence of SEQ ID NO:40, the FR3 sequence of SEQ ID NO:41, and the FR4 sequence of SEQ ID NO: 42. In other embodiments, the anti-complex I antibody comprises the HVRs of any of the embodiments described above, and further comprises a VL comprising the FR1 sequence of SEQ ID NO:43, the FR2 sequence of SEQ ID NO:44, the FR3 sequence of SEQ ID NO:41, and the FR4 sequence of SEQ ID NO: 42.

In any of the above antibodies, the VL FR3 sequence may be replaced by one selected from SEQ ID NO:67 or SEQ ID NO: 68.

In another aspect, an anti-complex I antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 46 or SEQ ID NO. 47. In particular embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-complex I antibody comprising that sequence retains the ability to bind to complex I. In particular embodiments, a total of 1-10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO 45, or SEQ ID NO 46, or SEQ ID NO 47. In particular embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in FRs). In particular embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:6, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:7, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8.

In another aspect, anti-complex I antibodies are provided, wherein the antibodies comprise a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 48 or SEQ ID NO. 49. In particular embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-complex I antibody comprising that sequence retains the ability to bind to complex I. In particular embodiments, a total of 1-10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO 48 or SEQ ID NO 49. In particular embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in FRs). In particular embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 9; (b) HVR-L2 comprising an amino acid sequence selected from SEQ ID NOs: 10-19; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In another aspect, there is provided an anti-complex I antibody, wherein said antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH of SEQ ID NO:45 and the VL sequence of SEQ ID NO:49, including post-translational modifications of these sequences. In one embodiment, the antibody comprises the VH of SEQ ID NO. 46 and the VL sequence of SEQ ID NO. 49, including post-translational modifications of these sequences. In another embodiment, the antibody comprises the VH of SEQ ID NO. 47 and the VL sequence of SEQ ID NO. 49, including post-translational modifications of these sequences. In another embodiment, the antibody comprises the VH of SEQ ID NO:45 and the VL sequence of SEQ ID NO:48, including post-translational modifications of these sequences. In another embodiment, the antibody comprises the VH of SEQ ID NO. 46 and the VL sequence of SEQ ID NO. 48, including post-translational modifications of these sequences. In another embodiment, the antibody comprises the VH of SEQ ID NO. 47 and the VL sequence of SEQ ID NO. 48, including post-translational modifications of these sequences.

In a further aspect, the invention provides antibodies that compete for and/or bind to the same epitope as the anti-complex I antibodies provided herein. For example, in particular embodiments, antibodies are provided that compete for and/or bind the same epitope as an anti-complex I antibody comprising a VH comprising the amino acid sequence of SEQ ID NOs:45-47 and a VL comprising the amino acid sequence of SEQ ID NO:48 or SEQ ID NO: 49.

In a further aspect, the invention provides an antibody that binds to the same epitope as an anti-complex I antibody comprising amino acids corresponding to amino acids selected from the group consisting of: a glutamine at position 47 of SEQ ID NO:203, a lysine at position 51 of SEQ ID NO:203, an aspartic acid at position 46 of SEQ ID NO:203, and combinations thereof. The corresponding amino acids comprising the epitope may be located at about the same position in the UL131 amino acid sequence, but differ due to differences in the amino acid sequence on UL131 between HCMV strains.

In a further aspect, the invention provides an antibody that binds to a polypeptide of HCMV complex I, wherein the polypeptide comprises amino acid sequence SRALPDQTRYKYVEQLVDLTLNYHYDAS (SEQ ID NO: 194).

In a further aspect, the invention provides antibodies that bind to the same epitope as the anti-complex I antibodies provided herein. In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-complex I antibody provided herein with an EC90 of 0.7. mu.g/ml, 0.5. mu.g/ml, 0.3. mu.g/ml, 0.1. mu.g/ml, 0.09. mu.g/ml, 0.08. mu.g/ml, 0.07. mu.g/ml, 0.06. mu.g/ml, 0.05. mu.g/ml, 0.04. mu.g/ml, 0.03. mu.g/ml, 0.02. mu.g/ml, 0.015, 0.012. mu.g/ml, 0.011. mu.g/ml, 0.010. mu.g/ml or less. In other aspects, the invention provides antibodies that bind the same epitope as the anti-complex I antibodies provided herein and at an antibody concentration of 0.05 μ g/ml, 0.02 μ g/ml, 0.015 μ g/ml, 0.014 μ g/ml, 0.013 μ g/ml, 0.012 μ g/ml, 0.011 μ g/ml, 0.010 μ g/ml, 0.009 μ g/ml, 0.008 μ g/ml, 0.007 μ g/ml, 0.006 μ g/ml, 0.005 μ g/ml, 0.004 μ g/ml, 0.003 μ g/ml, 0.002 μ g/ml, 0.001 μ g/ml, 0.0009 μ g/ml, 0.0008 μ g/ml, 0.0007 μ g/ml or less (e.g., at 10 μ g/ml, 0.015 μ g/ml, 0.007 μ g/ml, 0.006 μ g/ml, or less)^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M or lower antibody concentration) and 50% HCMV.

In bookIn a further aspect of the invention, the anti-complex I antibody according to any of the embodiments above is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, the anti-complex I antibody is an antibody fragment, such as Fv, Fab ', scFv, diabody, or F (ab')₂And (3) fragment. In another embodiment, the antibody is a full length antibody, e.g., a complete IgG1 antibody or other antibody species or isotype as defined herein.

In a further aspect, an anti-complex I antibody according to any of the embodiments above may incorporate any single feature or any combination thereof as described in sections 1-7 below.

B. Exemplary anti-gH antibodies

In one aspect, the invention provides an isolated antibody that binds gH. In particular embodiments, the anti-gH antibody specifically binds to an epitope of gH and neutralizes HCMV with an EC90 of 0.8 μ g/ml, 0.7 μ g/ml, 0.6 μ g/ml, 0.5 μ g/ml, 0.4 μ g/ml, 0.3 μ g/ml, 0.2 μ g/ml, 0.1 μ g/ml, 0.09 μ g/ml, 0.08 μ g/ml, 0.07 μ g/ml, 0.06 μ g/ml, 0.05 μ g/ml, 0.04 μ g/ml, 0.03 μ g/ml, 0.02 μ g/ml, 0.01 μ g/ml, 0.015, 0.010 μ g/ml or less. In some embodiments, the anti-gH antibody specifically binds to an epitope of the gH/gL dimer produced in baculovirus with an IC50 in the range of 0.01-0.17 nM. In various embodiments, the IC50 can be 0.01nM, 0.02nM, 0.03nM, 0.04nM, 0.05nM, 0.06nM, 0.07nM, 0.08nM, 0.09nM, 0.1nM, 0.11nM, 0.12nM, 0.13nM, 0.14nM, 0.15nM, 0.16nM, or 0.17 nM.

In other embodiments, the antibody binds to gH on the surface of HCMV and at an antibody concentration of 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.015, 0.014, 0.013, 0.012, 0.011, 0.010, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002 or less (e.g., at a concentration of 10. mu.g/ml, e.g., 10. mu.g/ml)^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M or lower antibody concentration) and 50% HCMV.

In one aspect, the invention provides anti-gH antibodies comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 71; (b) HVR-H2 comprising an amino acid sequence selected from SEQ ID NO 72, 73 or 74; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 75; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 76; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 77; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 78.

In one embodiment, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 71; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 72; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75. In another embodiment, the antibody comprises at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 71; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 73; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75. In another embodiment, the antibody comprises at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 71; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO 74; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75.

In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 76; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 77; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:78, and HVR-H2 comprising an amino acid sequence selected from SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO: 74.

In another aspect, the invention provides an antibody comprising (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:71, (ii) HVR-H2 comprising the amino acid sequence selected from SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74, and (iii) HVR-H3 comprising the amino acid sequence selected from SEQ ID NO:75, said VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:76, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:77, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 78.

In another aspect, the invention provides an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 71; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 72; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 75; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 76; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 77; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 78.

In another aspect, the invention provides an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 71; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 73; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 75; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 76; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 77; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 78.

In another aspect, the invention provides an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO 71; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO 74; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 75; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 76; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 77; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 78.

In particular embodiments, at any one or more amino acid substitutions in the anti-gH antibody as provided above occur at the following HVR positions: in HVR-H2 (SEQ ID NO: 91): positions 6 and 8. In particular embodiments, the substitutions are conservative substitutions, as provided herein. In particular embodiments, any one or more of the following substitutions may be made in any combination: in HVR-H2 (SEQ ID NO: 91): D6S, D6T, D6N, D6Q, D6F, D6M, D6L, and T8R. 93 covers all possible combinations of the above substitutions.

In any of the embodiments above, the anti-gH antibody is humanized. In one embodiment, the anti-gH antibody comprises HVRs as in any of the embodiments above, and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-gH antibody comprises HVRs as in any of the embodiments above, and further comprises a VH comprising the FR1 sequence of SEQ ID NO:79, the FR2 sequence of SEQ ID NO:80, the FR3 sequence of SEQ ID NO:81, and the FR4 sequence of SEQ ID NO: 82. In other embodiments, the anti-gH antibody comprises HVRs as in any of the embodiments described above, and further comprises a VL comprising the FR1 sequence of SEQ ID NO:83, the FR2 sequence of SEQ ID NO:84, the FR3 sequence of SEQ ID NO:85, and the FR4 sequence of SEQ ID NO: 86.

In another aspect, an anti-gH antibody of the invention comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ id no:92 but wherein the amino acid at position 54 is asn (n) and/or wherein the amino acid at position 56 is asn (r). In particular embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-gH antibody comprising that sequence retains the ability to bind gH. In a particular embodiment substitutions, insertions and/or deletions of a total of 1 to 10 amino acids have occurred in the sequence of SEQ ID NO 92 but in which the amino acid at position 54 is Asn (N) and/or in which the amino acid at position 56 is Asn (R). In particular embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in FRs). Optionally, the anti-gH antibody may comprise SEQ ID NO: 87. the VH sequence of SEQ ID NO:88 or SEQ ID NO:89, including post-translational modifications of the sequence. In particular embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:71, (b) HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 75.

In another aspect, an anti-gH antibody of the invention comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 90. In particular embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-gH antibody comprising that sequence retains the ability to bind gH. In a particular embodiment, substitutions, insertions and/or deletions of a total of 1 to 10 amino acids have occurred in SEQ ID NO: 90. In particular embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., in FRs). Optionally, the anti-gH antibody comprises the VL sequence of SEQ ID No. 90, including post-translational modifications of this sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 76; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 77; and (c) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 78.

In another aspect, the gH antibody of the invention comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO:87 and the VL sequence of SEQ ID NO:90, including post-translational modifications of these sequences.

In another embodiment, the antibody comprises the VH sequence of SEQ ID NO:88 and the VL sequence of SEQ ID NO:90, including post-translational modifications of these sequences.

In another embodiment, the antibody comprises the VH sequence of SEQ ID NO. 89 and the VL sequence of SEQ ID NO. 90, including post-translational modifications of these sequences.

In a further aspect, the invention provides antibodies that compete for and/or bind to the same epitope as the anti-gH antibodies provided herein. For example, in particular embodiments, antibodies are provided that compete for and/or bind the same epitope as an anti-gH antibody that includes a VH comprising the amino acid sequence of SEQ ID NOs:87, 88, or 89 and a VL comprising the amino acid sequence of SEQ ID NO: 90.

In a further aspect, the invention provides an antibody that binds to the same epitope as an anti-gH antibody comprising amino acids corresponding to amino acids selected from the group consisting of: tryptophan at position 168 of SEQ ID NO. 1, aspartic acid at position 446 of SEQ ID NO. 1; proline at position 171 of SEQ ID NO. 1, and combinations thereof. The corresponding amino acids comprising the epitope in the gH amino acid sequence may be located at about the same position, but may differ due to differences in the amino acid sequence in gH between individual HCMV strains.

In a further aspect, the invention provides antibodies that bind the same epitope as the anti-gH antibodies provided herein with an IC50 in the range of 0.01-0.17 nM. In various embodiments, IC50 may be 0.17nM or less (e.g., 0.16nM, 0.15nM, 0.14nM, 0.13nM, 0.12nM, 0.11nM, 0.10nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM, 0.01nM or less.) for example, in certain embodiments, an antibody that binds to the same epitope as HB1 (an anti-gH antibody comprising the VH sequence of SEQ ID NO:89 and the VL sequence of SEQ ID NO: 90) and has 0.17nM or less (e.g., 0.16nM, 0.15nM, 0.14nM, 0.13nM, 0.12nM, 0.11nM, 0.10nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04, 0.01nM, 0.5. mu.5. mu.8 g/nM, 0.5. mu.8 g/ml/nM, 0.5. mu.5 g/ml of IC/nM, 0.5 nM/ml/nM or less, EC90 of 0.1. mu.g/ml, 0.09. mu.g/ml, 0.08. mu.g/ml, 0.07. mu.g/ml, 0.06. mu.g/ml, 0.05. mu.g/ml, 0.04. mu.g/ml, 0.03. mu.g/ml, 0.02. mu.g/ml, 0.01. mu.g/ml, 0.015, 0.010. mu.g/ml or less neutralizes HCMV infection.

In other aspects, the invention provides antibodies that bind the same epitope as the anti-gH antibodies provided herein and are at a concentration of 0.1 μ g/ml, 0.09 μ g/ml, 0.08 μ g/ml, 0.07 μ g/ml, 0.06 μ g/ml, 0.05 μ g/ml, 0.04 μ g/ml, 0.03 μ g/ml, 0.02 μ g/ml, 0.015 μ g/ml, 0.014 μ g/ml, 0.013 μ g/ml, 0.012 μ g/ml, 0.011 μ g/ml, 0.010 μ g/ml, 0.009 μ g/ml, 0.008 μ g/ml, 0.007 μ g/ml, 0.006 μ g/ml, 0.005 μ g/ml, 0.004 μ g/ml, 0.003 μ g/ml, 0.002 μ g/ml, 0.001 μ g/ml, or less of the antibody (e.g/ml is at a concentration of 0.10 μ g/ml, e.^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M or lower antibody concentration) and 50% HCMV.

In a further aspect of the invention, the anti-gH antibody according to any of the embodiments above is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, the anti-gH antibody is an antibody fragment, such as Fv, Fab ', scFv, diabody, or F (ab')₂And (3) fragment. In another embodiment, the antibody is a full length antibody, e.g., a complete IgG1 antibody or other antibody species or isotype as defined herein.

In a further aspect, an anti-gH antibody according to the embodiments above may incorporate any single feature or any combination thereof as described in sections 1-7 below.

1. Affinity of antibody

In particular embodiments, an antibody of the invention as provided herein has ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (Kd).

In one embodiment, Kd is measured by performing a radiolabeled antigen binding assay (RIA) with the Fab form of the antibody of interest and its antigen, as described by the assay assays described below. By using a minimum concentration of (in the presence of a titration series of unlabelled antigen¹²⁵I) The solution binding affinity of Fab for antigen was measured by equilibration of the Fab with labeled antigen followed by capture of the bound antigen with an anti-Fab antibody coated plate (see, e.g., Chen et al, J.mol.biol.293:865-881 (1999)). In order to establish the conditions for the determination test,multi-well plates (Thermoscientific) were coated overnight with 5. mu.g/ml capture anti-Fab antibody (Cappel Labs) in 50mM sodium carbonate (pH 9.6) and subsequently blocked with 2% (w/v) bovine serum albumin in PBS at room temperature (about 23 ℃) for two to five hours. In the non-adsorption plate (Nunc # 269620), 100pM or 26pM [ alpha ], [ beta ]¹²⁵I]The antigen is mixed with serially diluted Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibodies, Fab-12, in Presta et al, Cancer Res.57: 4593-. The Fab of interest was then incubated overnight; however, incubation may continue for a longer period of time (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution was then removed and the plate was plated with 0.1% polysorbate 20 in PBSAnd washing eight times. When the plates had dried, 150. mu.l/well of scintillator (MICROSCINT-20) was added^TM(ii) a Packard) and in TOPCOUNT^TMThe plate was counted on a gamma counter (Packard) for ten minutes. The concentration of each Fab that gives less than or equal to 20% maximal binding is selected for use in a competition binding assayThe application is as follows.

According to another embodiment, at 25 ℃ use is made of-2000 or(BIAcore, inc., Piscataway, NJ), using an immobilized antigen CM5 chip, with-10 Response Units (RU) using surface plasmon resonance measurements, to measure Kd. Briefly, carboxymethylated dextran biosensor chips (CM 5, BIACORE, Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Before injecting the antigen at a flow rate of 5. mu.l/min, the antigen was diluted to 5. mu.g/ml (. about.0.2. mu.M) with 10mM sodium acetate, pH4.8, to achieve about 10 Response Units (RU) of conjugated protein. After antigen injection, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, at a flow rate of about 25. mu.l/min, at 25 ℃, with 0.05% polysorbate 20 (TWEEN-20)^TM) Surfactant in PBS (PBST) two-fold serial dilutions (0.78 nM-500 nM) of Fab were injected. (ii) use of a simple one-to-one Langmuir binding model by simultaneous fitting of binding and dissociation sensorgrams: (Evaluation Software version 3.2), calculate the association rate (k)_on) And dissociation rate (k)_off). Equilibrium dissociation constant (Kd) was calculated as k_off/k_onA ratio. See, e.g., Chen et al, J.mol.biol.293:865-881 (1999). If the binding rate is more than 10 as determined by surface plasmon resonance as described above⁶M^-1s^-1The binding rate can then be determined by using a fluorescence quenching technique that measures binding at increasing concentrationsIncrease or decrease in fluorescence emission intensity of 20nM anti-antigen antibody (Fab form) in PBS, pH7.2 at 25 ℃ (excitation =295 nM; emission =340nM, 16nM bandpass) in the presence of a degree of antigen, as in a spectrophotometer such as that equipped with a flow stopping device (Aviv Instruments) or 8000 series SLM-AMINCO with a stirred cuvette^TMMeasured in a spectrophotometer (thermospectonic).

2. Antibody fragments

In particular embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab '-SH, F (ab') 2, Fv, and scFv fragments and others described below. For a review of some antibody fragments see Hudson et al nat. Med.9: 129-. For reviews on scFv fragments see, for example, Pluckth ü n, The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore eds (Springer-Verlag, New York), p.269-315 (1994); see also WO 93/16185; and U.S. patent nos. 5,571,894 and 5,587,458. Relating to Fab and F (ab') containing a salvage receptor (salvaging receptor) binding epitope residue and having increased in vivo half-life₂See U.S. Pat. No. 5,869,046 for a discussion of fragments.

Diabodies are antibody fragments with two antigen binding sites and may be bivalent or bispecific. See, for example, EP404,097; WO 1993/01161; hudson et al, nat. Med.9: 129-; and Hollinger et al, proc.natl.acad.sci.usa90: 6444-6448(1993). Tri-antibodies (Triabody) and tetra-antibodies (Tetrabody) are also described in Hudson et al, nat. Med.9:129-134 (2003).

A single domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In particular embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B 1).

Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., e.coli or phage), as described herein.

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Some chimeric antibodies are described, for example, in U.S. Pat. nos. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In yet another example, a chimeric antibody is a "class-switched" antibody, wherein the class or subclass of the antibody has been different from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In particular embodiments, the chimeric antibody is a humanized antibody. Generally, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies comprise one or more variable domains in which HVRs, such as CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody optionally may further comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., an antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for making the same are reviewed, for example, in Almagro and Fransson, front.biosci.13:1619-1633 (2008), and further described, for example, in the following references: riechmann et al, Nature332: 323-E329 (1988); queen et al, Proc.nat' l Acad.Sci.USA86:10029-10033 (1989); U.S. patent nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; kashmiri et al, Methods36:25-34 (2005) (describing SDR (a-CDR) grafting); padlan, mol.Immunol.28:489-498 (1991) (description "resurfacing"); dall' Acqua et al, Methods36:43-60 (2005) (description "FR shuffling"); and Osbourn et al, Methods36:61-68 (2005) and Klimka et al, Br.J. cancer, 83:252-260 (2000) (describing the "guided selection" method of FR shuffling).

Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best-fit" (best-fit) method (see, e.g., Sims et al J.Immunol.151:2296 (1993)); framework regions derived from human antibody consensus sequences for a particular subset of light or heavy chain variable regions (see, e.g., Carter et al Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al J.Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front.biosci.13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al, J.biol. chem.272:10678-10684 (1997) and Rosok et al, J.biol. chem.271:22611-22618 (1996)).

4. Human antibodies

In particular embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are described in van Dijk and van de Winkel, curr. 368-74 (2001) and Lonberg, curr. Opin. Immunol.20: 450-.

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus, either replacing an endogenous immunoglobulin locus, or present extrachromosomally or randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin genome has typically been inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, e.g., the description of XENOMOUSE^TMU.S. Pat. nos. 6,075,181 and 6,150,584 to technology; description of the technologyUs patent numbers 5,770,429; description of K-MU.S. Pat. No. 7,041,870 to Art, and descriptionU.S. patent application publication No. US2007/0061900 of the art). The human variable regions from the whole antibodies produced by such animals may be further modified, for example by combination with different human constant regions.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for human monoclonal antibody production have been described. (see, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al, Monoclonal antibody production Techniques and Applications, pp 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J.Immunol., 147: 86 (1991)), human antibodies generated via human B-cell hybridoma technology are also described in Li et al, Proc.Natl.Acad.Sci.USA, 103: 3557-. Additional methods include, for example, those described in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26 (4): 265-268 (2006) (describing human-human hybridomas). The human hybridoma technique (Trioma technique) is also described in Vollmers and Brandlens, Histology and Histopathology, 20 (3): 927-.

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

5. Library-derived antibodies

The antibodies in the compositions of the invention can be isolated by screening combinatorial libraries for antibodies having one or more desired activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies having desired binding characteristics. Such Methods are reviewed, for example, in Hoogenboom et al in Methods in Molecular Biology178:1-37 (O' Brien et al eds., Human Press, Totowa, NJ, 2001), and are further described, for example, in the following documents: McCafferty et al, Nature348: 552-554; clackson et al, Nature352:624-628 (1991); marks et al, j.mol.biol.222: 581-597 (1992); marks and Bradbury, Methods in Molecular Biology248:161-175 (Lo, eds., Human Press, Totowa, NJ, 2003); sidhu et al, j.mol.biol.338 (2): 299-310 (2004); lee et al, j.mol.biol.340 (5): 1073-1093 (2004); fellouse, proc.natl.acad.sci.usa101 (34): 12467-12472 (2004); and Lee et al, j.immunol.methods284 (1-2): 119-132(2004).

In some phage display methods, a repertoire of VH and VL genes (reporters) is cloned separately by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages, such as Winter et al, ann. 433 and 455 (1994). Phage typically display antibody fragments, either as single chain fv (scFv) fragments or Fab fragments. Libraries from immunized sources can provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive pools may be cloned () (e.g. from humans) to provide a solution for a wide range of non-self and selfA single antibody source of antigen without any immunization, as described by Griffiths et al, EMBOJ, 12: 725-734 (1993). Finally, naive libraries can also be prepared synthetically by: unrearranged V gene segments are cloned from stem cells, PCR primers containing random sequences are used to encode the highly variable CDR3 regions and rearrangement is accomplished in vitro, as described by Hoogenboom and Winter, j.mol.biol., 227: 381 and 388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373 and U.S. patent publication nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360.

Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein.

6. Multispecific antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies with binding specificities directed against at least two different sites. In particular embodiments, one of the binding specificities is for complex I or gH, and the other is for any other antigen. In particular embodiments, one of the binding specificities is for complex I and the other is for gH. In particular embodiments, the bispecific antibody can bind to two different epitopes of complex I or gH. Bispecific antibodies can also be used to localize cytotoxic agents to cells having complex I or gH on the cell surface. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO93/08829 and traumecker et al, EMBO j.10: 3655 (1991)), and "knob-in-hole" (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by: engineering electrostatic steering effects (electrostatic steering effects) to produce antibody Fc-heterodimer molecules (WO 2009/089004a 1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al, Science, 229: 81 (1985)); use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al, J.Immunol., 148 (5): 1547-1553 (1992)); the "diabody" technique was used to prepare bispecific antibody fragments (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA, 90: 6444-; and the use of single chain fv (sFv) dimers (see, e.g., Gruber et al, J.Immunol., 152:5368 (1994)); and e.g. Tutt et al j.immunol.147: 60 (1991) the trispecific antibody is prepared as described.

Engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, e.g., US2006/0025576a 1).

Antibodies or fragments also include herein "Dual action fabs" (Dual activating fabs) or "DAFs" comprising an antigen binding site that binds to complex I or gH and another different antigen (see, e.g., US 2008/0069820).

7. Antibody variants

In particular embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

a) Substitution, insertion and deletion variants

In particular embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in table 1 under the heading "conservative substitutions". More substantial changes are provided under the heading "exemplary substitutions" of table 1 and are further described below with reference to amino acid side chain species. Amino acid substitutions may be introduced into the antibody of interest and the product screened for a desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

TABLE 1

Amino acids can be grouped according to common side chain properties:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: cys, Ser, Thr, Asn, Gln;

(3) acidic: asp, Glu;

(4) basic: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions require the exchange of a member of one of these classes for another.

One class of substitutional variants involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have alterations (e.g., improvements) in some biological properties relative to the parent antibody (e.g., increased affinity, decreased immunogenicity), and/or will substantially retain some of the biological properties of the parent antibody. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, e.g., using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated, and variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Alterations (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such alterations can be made in HVR "hot spots" (i.e., residues encoded by codons that are mutated at high frequency during somatic maturation (see, e.g., Chowdhury, Methods mol. biol.207: 179. 196 (2008))), and/or SDRs (a-CDRs), and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and re-selection from secondary libraries has been described, for example, in Hoogenboom et al in Methods in Molecular Biology178:1-37 (O' Brien et al, eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Secondary libraries were then constructed. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves HVR-directed methods in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine screening mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly commonly targeted.

In particular embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in HVRs. Such changes may be outside of HVR "hotspots" or SDRs. In particular embodiments of the variant VH and VL sequences provided above, each HVR is either unaltered or contains no more than one, two or three amino acid substitutions.

One useful method for identifying antibody residues or regions that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether antibody interaction with an antigen is affected. Further substitutions may be introduced at amino acid positions demonstrating functional sensitivity to the initial substitution. Alternatively or additionally, the contact points between the antibody and the antigen are identified by the crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues may be targeted or eliminated as candidates for substitution. Variants can be screened to determine if they contain the desired property.

Amino acid sequence insertions include amino and/or carboxy terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion of an enzyme (e.g., for ADEPT) or polypeptide that increases the serum half-life of the antibody to the N-or C-terminus of the antibody.

b) Glycosylation variants

In particular embodiments, the antibodies provided herein are altered to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of glycosylation sites to the antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

When the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides, which are typically N-linked to Asn297 of the CH2 domain attached to the Fc region. See, e.g., Wright et al TIBTECH15:26-32 (1997). Oligosaccharides may include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the "stem" of a biantennary oligosaccharide structure. In some embodiments, modifications of oligosaccharides may be made in the antibodies of the invention in order to produce antibody variants with specific improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure that lacks (directly or indirectly) fucose attached to an Fc region. For example, the amount of fucose in such antibodies may be 1% -80%, 1% -65%, 5% -65%, or 20% -40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain of Asn297, as measured by MALDI-TOF mass spectrometry, relative to the sum of all sugar structures (e.g. complex, hybrid and high mannose structures) attached to Asn 297. Asn297 refers to the asparagine residue at about position 297 of the Fc region (Eu numbering of Fc region residues); however, due to minor sequence variations in the antibody, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, e.g., U.S. patent publication No. US2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; okazaki et al J.mol.biol.336:1239-1249 (2004); Yamane-Ohnuki et al Biotech.Bioeng.87: 614(2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13CHO cells deficient in protein fucosylation (Ripka et al Arch. biochem. Biophys.249:533-545 (1986); U.S. patent application Ser. No. US2003/0157108A1, Presta, L; and WO2004/056312A1, Adams et al, particularly on example 11), and knock-out cell lines, such as the α -1, 6-fucosyltransferase gene, FUT8, knock-out CHO cells (see, e.g., Yamane-Ohnuki et al Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al, Biotechnol. Bioeng., 94 (4): 680-688 (2006); and WO 2003/085107).

Further provided are antibody variants having bisected oligosaccharides (biantennary oligosaccharides), for example, wherein biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003/011878 (Jean-Mairet et al); U.S. Pat. No. 6,602,684 (Umana et al); and US2005/0123546 (Umana et al). Also provided are antibody variants having at least one galactose residue in an oligosaccharide attached to an Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997/30087 (Patel et al); WO1998/58964 (Raju, S.); and WO1999/22764 (Raju, S.).

c) Fc region variants

In particular embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. Fc region variants may include human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4Fc regions) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions.

In particular embodiments, the invention contemplates antibody variants with some, but not all, effector functions, which make them desirable candidates for use in applications where the half-life of the antibody in vivo is important, while some effector functions (e.g., complement and ADCC) are not required or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to demonstrate the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assay assays may be performed to ensure that antibodies lack fcyr binding (and thus may lack it)Lack ADCC activity) but retain FcRn binding ability. The main cell for mediating ADCC, NK cell, expresses onlyAnd monocytes are expressedAndFcR expression on hematopoietic cells is summarized in Table 3 on page 464 of ravatch and Kinet, Annu.Rev.Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of molecules of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al Proc. nat 'l Acad. Sci. USA83:7059-7063 (1986)) and Hellstrom, I. et al, Proc. nat' l Acad. Sci. USA82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al, J.Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI for flow cytometry)^TMNon-radioactive cytotoxicity assay (Celltechnology, Inc. mountain View, CA; andnon-radioactive cytotoxicity assay (Promega, Madison, WI). Effector cells that can be used in such assay include Peripheral Blood Mononuclear Cells (PBMCs) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model, for example as disclosed in Clynes et al Proc. nat' l Acad. Sci. USA95: 652-. A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., C1q and C3C binding ELISAs in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, e.g., forGazzano-Santoro et al, J.Immunol.Methods202:163 (1996); cragg, M.S. et al, Blood101:1045-1052 (2003); and Cragg, M.S. and M.J.Glennie, Blood103: 2738-. FcRn binding and in vivo clearance/half-life determinations can also be made using methods known in the art (see, e.g., Petkova, s.b. et al, Int' l.immunol.18 (12): 1759-.

Antibodies with reduced effector function include those with substitutions of one or more of residues 238, 265, 269, 270, 297, 327 and 329 of the Fc region (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants having substitutions of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

Specific antibody variants with improved or reduced FcRs binding are described. (see, e.g., U.S. Pat. No. 6,737,056; WO2004/056312 and Shields et al, J.biol.chem.9 (2): 6591-6604 (2001))

In particular embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, for example substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In some embodiments, alterations are made in the Fc region to result in altered (e.g., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. nos. 6,194,551, WO99/51642, and Idusogie et al j.immunol.164: 4178 4184 (2000).

Antibodies with increased half-life and improved binding of the neonatal Fc receptor (FcRn) responsible for the transfer of maternal IgGs to the fetus are described in US2005/0014934A1 (Hinton et al) (Guyer et al, J.Immunol.117:587 (1976) and Kim et al, J.Immunol.24:249 (1994)). These antibodies comprise an Fc region having one or more substitutions therein, which improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238. 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, by substitution of residue 434 of the Fc region (U.S. patent No. 7,371,826).

For additional examples of Fc region variants, see also Duncan & Winter, Nature322:738-40 (1988); U.S. Pat. nos. 5,648,260; U.S. Pat. nos. 5,624,821; and WO 94/29351.

d) Cysteine engineered antibody variants

In particular embodiments, it may be desirable to prepare cysteine engineered antibodies, such as "thioMAbs," in which one or more residues of the antibody are replaced with cysteine residues. In particular embodiments, the residues that are replaced are located at accessible sites on the antibody. By replacing these residues with cysteine, reactive sulfhydryl groups are placed on accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as a drug moiety or a linker-drug moiety, to produce an immunoconjugate, as further described herein. In particular embodiments, any one or more of the following residues may be substituted with a cysteine residue: v205 of the light chain (Kabat numbering); a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antibodies can be generated as described, for example, in U.S. patent No. 7,521,541.

e) Antibody derivatives

In particular embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, polyethylene,Poly-1, 3-dioxolane, poly-1, 3, 6-trisAlkanes, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polyoxypropylene/polyoxyethylene copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde, because of its stability in water, may have advantages in manufacturing. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative is used in therapy under defined conditions, and the like.

In another embodiment, conjugates of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. USA 102: 11600-. The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm normal cells, but heat the non-proteinaceous moiety to a temperature that can kill cells in the vicinity of the antibody-non-proteinaceous moiety.

C. Recombinant methods and compositions

Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, isolated nucleic acids encoding an anti-complex I antibody or an anti-gH antibody described herein are provided. The nucleic acid may encode an amino acid sequence comprising a VL of an antibody and/or an amino acid sequence comprising a VH of an antibody (e.g., a light and/or heavy chain of an antibody). In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and an amino acid sequence comprising an antibody VH, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH. In one embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an anti-complex I antibody or an anti-gH antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of anti-complex I antibodies or anti-gH antibodies, antibody-encoding nucleic acids, e.g., as described above, are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. This nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237, 5,789,199, and 5,840,523. (see also Charlton, Methods in molecular Biology, Vol.248 (B.K.C.Lo, eds., Humana Press, Totowa, NJ, 2003), p.245-254, describing the expression of antibody fragments in E.coli). After expression, the antibody can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized", which results in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. Biotech.22: 1409-.

Suitable host cells for expression of glycosylated antibodies may also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified which can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIIES for antibody production in transgenic plants^TMA technique).

Vertebrate cells can also be used as hosts. For example, suspension growth adapted mammalian cell lines may be useful. Other examples of useful mammalian host cell lines are SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (such as for example Graham et al, J.Gen Virol.36:59 (1977) in the 293 or 293 cell); baby hamster kidney cells (BHK); mouse Sertoli cells (such as, for example, TM4 cells as described in Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumors (MMT 060562); TRI cells as described, for example, in Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982); MRC5 cells; and FS4 cells other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc.Natl.Acad.Sci.USA77:4216 (1980)), and myeloma cell lines such as Y0, NS0 and Sp 2/0. for reviews of some mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in biological, Vol.K.248, Lowman.C.255, Huwa, edited, Vol.J., Prewa, Vol.268).

D. Determination test

The anti-complex I antibodies or anti-gH antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assay assays known in the art.

1. Binding assay and other assay assays

In one aspect, the antibodies of the invention are tested for their antigen binding activity by known methods, such as ELISA, western blot, and the like.

In another aspect, a competition assay can be used to identify antibodies that compete with the anti-complex I antibodies described herein for binding to complex I.

In another aspect, a competition assay can be used to identify antibodies that compete with anti-gH antibodies described herein for binding gH.

In particular embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) as gH or complex I.

Detailed exemplary Methods for Mapping the Epitope bound by an antibody are provided in Morris (1996) "Epitope Mapping Protocols," Methods in Molecular Biology Vol.66 (Humana Press, Totowa, NJ).

In an exemplary competition assay, immobilized complex I or gH is incubated in a solution comprising a first labeled antibody that binds to complex I or gH, and a second unlabeled antibody to be tested for its ability to compete with the first antibody for binding to complex I or gH. The second antibody may be present in the hybridoma supernatant. As a control, immobilized complex I or gH is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the primary antibody to bind to complex I or gH, excess unbound antibody is removed and the amount of label bound to immobilized complex I or gH is measured. If the amount of label bound to immobilized complex I or gH is substantially reduced in the test sample relative to the control sample, this indicates that the second antibody competes with the first antibody for binding to complex I or gH. See Harlow and Lane (1988) Antibodies: chapter 14 of A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Competition assay assays can also be performed with FACS in the manner described above, using cells transfected with gH and/or other members of complex I or complex II and expressed on the cell surface. In addition, ELISA using gH and/or reconstituted complex I or complex II can also be used in competition assay assays. The use of FACS and ELISA for measuring anti-gH and anti-complex I antibodies is further described in the examples.

2. Activity assay

In one aspect, an assay for identifying an anti-complex I antibody having biological activity is provided. Biological activity can include, for example, specific binding to a conformational epitope formed by the binding of UL128, UL130, UL131, and gH/gL, or specific binding to an epitope within a single protein of complex I, neutralizing HCMV with an EC90 of 0.7 μ g/ml or less. In some embodiments, EC90 is 0.5 μ g/ml or less. In other embodiments, EC90 is 0.3 μ g/ml or less. In other embodiments, EC90 is 0.1 μ g/ml or less. In other embodiments, EC90 is 0.08 μ g/ml or less. In other embodiments, EC90 is 0.06 μ g/ml or less. In still other embodiments, EC90 is 0.04 μ g/ml or less. In other embodiments, EC90 is 0.02 μ g/ml or less. In other embodiments, EC90 is 0.015. mu.g/ml or less. In other embodiments, EC90 is 0.012 μ g/ml or less. In other embodiments, the EC90 is 0.011 μ g/ml or less. In other embodiments, the EC90 is 0.010 μ g/ml or less. Compositions comprising antibodies having such biological activities are also provided.

In one aspect, an assay for identifying an anti-gH antibody having biological activity is provided. Biological activity can include, for example, neutralizing HCMV with an EC90 of 1. mu.g/ml, 0.9. mu.g/ml, 0.8. mu.g/ml, 0.7. mu.g/ml, 0.6. mu.g/ml, 0.5. mu.g/ml, 0.4. mu.g/ml, 0.3. mu.g/ml, 0.2. mu.g/ml, 0.1. mu.g/ml, 0.09. mu.g/ml, 0.08. mu.g/ml, 0.07. mu.g/ml, 0.06. mu.g/ml, 0.05. mu.g/ml, 0.04. mu.g/ml or less.

An anti-gH antibody binds gH/gL dimers expressed in baculovirus with an IC50 of 0.17nM or less (e.g., 0.16nM, 0.15nM, 0.14nM, 0.13nM, 0.12nM, 0.11nM, 0.10nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM, 0.01nM or less) in the composition of the invention. Also provided are compositions comprising antibodies having this biological activity in vivo and/or in vitro.

In particular embodiments, the antibodies of the invention are tested for this biological activity. For an exemplary description of such assay tests, see example 3.

E. Immunoconjugates

The invention also provides compositions comprising an immunoconjugate comprising the anti-complex I antibody or anti-gH antibody herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (maytansinoids) (see U.S. Pat. nos. 5,208,020, 5,416,064, and european patent EP0425235B 1); auristatins (auristatins) such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. patent nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin (dolastatin); calicheamicin (calicheamicin) or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al, Cancer Res.53:3336-3342 (1993); and Lode et al, Cancer Res.58:2925-2928 (1998)); anthracyclines such as daunomycin (daunomycin) or doxorubicin (see Kratz et al, Current Med. chem.13: 477-) (2006); Jeffrey et al, Bioorganic & Med. chem.Letters16: 358-; methotrexate; vindesine (vindesine); taxanes (taxanes) such as docetaxel (docetaxel), paclitaxel (paclitaxel), larotaxel, tesetaxel and ortataxel; trichothecene; and CC 1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria toxin a chain, a non-binding active fragment of diphtheria toxin, exotoxin a chain (from Pseudomonas aeruginosa), ricin a chain, abrin a chain, modeccin a chain, α -sarcin, eleostema orientalis (Aleurites fordii) protein, carnation protein, phytolacca americana (Phytolaca americana) protein (PAPI, PAPII and PAP-S), momordica charantia (momordia) inhibitor, curcin protein, crotin protein, grasses (saononacia officinalis) inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and trichothecene.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate.A variety of radioisotopes are available for the production of radioconjugates. Examples include At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu. When the radioconjugate is used for detection, it may contain a radioactive atom for scintillation studies, such as tc99m or I123, or a spin label for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron again.

Conjugates of the antibody and cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl 3- (2-pyridinedithiol) propionate (SPDP), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis (p-azidobenzoyl) hexanediamine), bis-diazoniumyl compoundsDerivatives (e.g. bis (para-diazo)Benzoyl) -ethylenediamine), diisocyanates (e.g., toluene 2, 6-diisocyanate), and bis-reactive fluorine compounds (e.g., 1, 5-difluoro-2, 4-dinitrobenzene). For example, the ricin immunotoxin may be as described in Vitetta et al Science 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugation of radionuclides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-sensitive linkers, peptidase-sensitive linkers, photosensitive linkers may be usedA dimethyl linker or a disulfide bond-containing linker (Chari et al Cancer Research 52: 127-131 (1992)).

Immunoconjugates or ADCs are specifically contemplated herein, but are not limited to, such conjugates prepared with cross-linking linker reagents, including but not limited to BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB and SVSB (succinimidyl- (4-vinylsulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, inc., Rockford, il., u.s.a.).

F. Methods and compositions for diagnosis and detection

In particular embodiments, any anti-complex I antibody and/or anti-gH antibody, or composition comprising such an antibody, as provided herein, can be used to detect the presence of complex I and/or gH in a biological sample. The term "detecting" as used herein encompasses quantitative or qualitative detection. In particular embodiments, the biological sample comprises cells or tissues, such as placenta, kidney, heart, lung, liver, pancreas, intestine, thymus, bone, tendon, cornea, skin, heart valves, and veins. In addition, compositions comprising the antibodies can be used to detect HCMV in endothelial cells, epithelial cells, fibroblasts, and macrophages.

In one embodiment, anti-complex I antibodies and/or anti-gH antibodies are provided for use in a method of diagnosis or detection. In a further aspect, a method of detecting the presence of complex I and/or gH in a biological sample is provided. In particular embodiments, the method comprises contacting the biological sample with an anti-complex I antibody and/or an anti-gH antibody as described herein under conditions that allow the anti-complex I antibody to bind to complex I and/or the anti-gH antibody to bind to gH, and detecting whether a complex between the anti-complex I antibody and complex I and/or the anti-gH antibody and gH is formed. The method may be an in vitro or in vivo method. In one embodiment, the anti-complex I antibody or anti-gH antibody, or the combination of anti-complex I antibody and anti-gH antibody, is used to select eligible subjects for therapy using the anti-complex I antibody or anti-gH antibody or the combination of anti-complex I antibody and anti-gH antibody, e.g., when complex I and gH are biomarkers for patient selection.

Exemplary conditions that can be diagnosed using the compositions of the invention include HCMV infection, e.g., HCMV infection from transplanted organs or tissues, congenital HCMV infection, HCMV infection during pregnancy, and HCMV infection in children, infants, and adults.

In particular embodiments, compositions comprising labeled anti-complex I antibodies and/or anti-gH antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties that are detected indirectly (e.g., by enzymatic reactions or molecular interactions), such as enzymes or ligands. Exemplary labels include, but are not limited to, radioisotopes³²P、¹⁴C、¹²⁵I、³H and¹³¹i, fluorophores such as rare earth chelates or luciferin and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidases such as glucose oxidase, galactose oxidase and 6-phosphoglucose dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with enzymes such as HRP, lactoperoxidase or microperoxidase that use hydrogen peroxide to oxidize dye precursors, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

G. Pharmaceutical preparation

Antibodies of the desired purity can be prepared by combining the antibody with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences16th edition, Osol,A. labeling (1980)) to prepare a pharmaceutical formulation of an anti-complex I antibody or an anti-gH antibody or a combination of an anti-complex I antibody and an anti-gH antibody as described herein, in a lyophilized formulation or in an aqueous solution. As described herein, the anti-complex I antibody and the anti-gH antibody can be formulated in a single combined pharmaceutical formulation or separate pharmaceutical formulations. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (e.g. octadecyl dimethyl benzyl ammonium chloride; chlorhexidine di-ammonium; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers further include interstitial (injectable) drug dispersants such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 (r: (r) ())Baxter International, Inc.). Some exemplary sHASEGPs and methods of use include rHuPH20, described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations including histidine-acetate buffers.

In addition to the anti-complex I antibody and/or anti-gH antibody, the formulations herein may also contain active ingredients required for the particular indication to be treated, preferably those having complementary activities that do not adversely affect each other. For example, it may be desirable to further provide ganciclovir, foscarnitin, valganciclovir, and silurovir. Such active ingredients are suitably present in the combination in an amount effective for the intended purpose.

The active ingredient may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's pharmaceutical sciences, 16th edition, Oslo, a., ed., (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices may be in the form of shaped articles, e.g., films, or microcapsules.

The formulations to be used for in vivo administration are generally sterile. Sterility can be readily achieved by filtration, for example, through sterile filtration membranes.

H. Therapeutic methods and compositions

Any composition comprising an anti-complex I antibody and/or an anti-gH antibody provided herein can be used in a method of treatment.

In one aspect, a composition comprising an anti-complex I antibody or an anti-gH antibody or an anti-complex I antibody and an anti-gH antibody is provided for use as a medicament. In a further aspect, there is provided a composition comprising an anti-complex I antibody or an anti-gH antibody or an anti-complex I antibody and an anti-gH antibody for use in the treatment of HCMV infection. In particular embodiments, compositions comprising an anti-complex I antibody or an anti-gH antibody or an anti-complex I antibody and an anti-gH antibody are provided for use in methods of treatment. In particular embodiments, the invention provides a composition comprising an anti-complex I antibody or an anti-gH antibody or an anti-complex I antibody and an anti-gH antibody for use in a method of treating a subject having an HCMV infection, the method comprising administering to the subject an effective amount of a composition comprising an anti-complex I antibody and/or an anti-gH antibody. In other embodiments, the invention provides compositions for use in a method of preventing, inhibiting, or treating a congenital HCMV infection or an HCMV infection in a tissue or organ transplant recipient for which the transplanted tissue, organ or donor is or has been infected with HCMV. In one such embodiment, the tissue or organ transplant recipient is seronegative for HCMV infection. In additional embodiments, the transplant recipient or individual has been previously infected with HCMV and is at risk for HCMV reactivation and infection. In certain embodiments, the method further comprises administering to the individual or transplant recipient an effective amount of at least one additional therapeutic agent, as described below. In other embodiments, the invention also provides a composition comprising an anti-complex I antibody or an anti-gH antibody or an anti-complex I antibody and an anti-gH antibody for use in a method of treatment of an infant infected with HCMV or an infant exposed to HCMV during pregnancy, the method of treatment comprising administering to the infant an effective amount of a composition comprising an antibody of the invention or a combination thereof. In a further embodiment, the invention provides a composition comprising an anti-complex I antibody or an anti-gH antibody or an anti-complex I antibody and an anti-gH antibody for use in treating, inhibiting or preventing HCMV infection in an individual at risk of infection. An "individual" according to any of the embodiments above is preferably a human.

In a further aspect, the invention provides the use of a composition comprising an anti-complex I antibody and/or an anti-gH antibody or an anti-complex I antibody and an anti-gH antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating, preventing or inhibiting HCMV infection. In a further embodiment, the medicament is for use in treating, preventing, or inhibiting HCMV infection, comprising administering to an individual having HCMV infection an effective amount of the medicament. In other embodiments, the medicament is for use in a method of preventing, inhibiting or treating a congenital HCMV infection or an HCMV infection in a tissue or organ transplant recipient for which the transplanted tissue, organ or donor is or has been infected with HCMV. In one such embodiment, the tissue or organ transplant recipient is seronegative for HCMV infection. In additional embodiments, the transplant recipient or individual has been previously infected with HCMV and is at risk for HCMV reactivation and infection. In particular embodiments, the medicament further comprises an effective amount of at least one additional therapeutic agent, e.g., as described below. In a further embodiment, the medicament is for treating, inhibiting, or preventing HCMV infection in an individual at risk of infection, comprising administering to the individual an amount of the medicament effective to inhibit or prevent HCMV infection. In other embodiments, the medicament is for treating an infant infected with HCMV or an infant exposed to HCMV during pregnancy comprising administering to the infant an effective amount of a composition comprising an antibody of the invention or a combination thereof. An "individual" according to any of the embodiments described above may be a human. In particular embodiments, the medicament is for reducing HCMV viral titer or preventing an increase in HCMV viral titer in an individual. In one embodiment, the method comprises administering to the individual an effective amount of a composition comprising an anti-complex I antibody and/or an anti-gH antibody to reduce HCMV viral titer or prevent an increase in HCMV viral titer. In one embodiment, an "individual" is a human and/or pregnant woman at risk for HCMV infection and/or an organ transplant recipient.

In a further aspect, the invention provides methods for treating, preventing or inhibiting HCMV infection. In one embodiment, the method comprises administering to the individual an effective amount of a composition comprising an anti-complex I antibody and/or an anti-gH antibody. In other embodiments, the invention provides methods of preventing, inhibiting, or treating a congenital HCMV infection or an HCMV infection in a tissue or organ transplant recipient for which the transplanted tissue, organ or donor is or has been infected with HCMV, comprising administering to the individual or transplant recipient an effective amount of a composition comprising an anti-complex I antibody and an anti-gH antibody. In one such embodiment, the tissue or organ transplant recipient is seronegative for HCMV infection. In additional embodiments, the transplant recipient or individual has been previously infected with HCMV and is at risk for HCMV reactivation and infection. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent as described below. In other embodiments, the invention provides methods for treating, inhibiting or preventing HCMV infection or exposure to HCMV during pregnancy comprising administering to an infant an effective amount of a composition comprising an antibody of the invention or a combination thereof. An "individual" according to any of the embodiments described above may be a human.

In a further aspect, the invention provides methods for inhibiting or preventing HCMV infection in an individual at risk of infection. In one embodiment, the method comprises administering to the individual an effective amount of a composition comprising an anti-complex I antibody and/or an anti-gH antibody to inhibit or prevent HCMV infection. In one embodiment, the "individual" is a human.

In particular embodiments, the invention provides methods for reducing HCMV viral titer or preventing an increase in HCMV viral titer in an individual. In one embodiment, the method comprises administering to the individual an effective amount of a composition comprising an anti-complex I antibody and/or an anti-gH antibody to reduce HCMV viral titer or prevent an increase in HCMV viral titer. In one embodiment, an "individual" is a human and/or pregnant woman at risk for HCMV infection and/or an organ transplant recipient.

HCMV viral titers can be measured by any method known in the art, e.g., by ELISA to measure viral antibodies, serological or histological-based assay by quantifying the amount of viral DNA in a sample (specific viral genes and/or viral genomes to determine viral load) and/or culturing viruses from a sample to measure the presence of HCMVAt this point. Such diagnostic tests are commercially available, e.g.CMV Test andAMPLICOR CMV MONITOR Test (Roche), which can be used to diagnose HCMV infection and MONITOR antiviral therapy by quantifying HCMV DNA. In particular embodiments, the viral titer of HCMV in a phase individual is reduced by any one of the following values, relative to an untreated individual or relative to the viral titer of the same individual prior to treatment: about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or less.

In further embodiments, the transplanted organ or tissue may be any organ or tissue capable of being transplanted from one individual to a second individual. For example, the transplanted organ may be, but is not limited to, heart, kidney, liver, lung, pancreas, intestine, or thymus. Additionally, for example, the transplanted tissue may be, but is not limited to, hand, cornea, skin, face, pancreatic islets, bone marrow, stem cells, whole blood, platelets, serum, blood cells, blood vessels, heart valves, bone progenitor cells, cartilage, ligaments, tendons, muscle linings.

In a further aspect, the invention provides compositions and pharmaceutical formulations comprising any of the anti-complex I antibodies and/or anti-gH antibodies provided herein, e.g., for use in any of the methods of treatment described above. In one embodiment, the pharmaceutical formulation comprises any of the anti-complex I antibodies and/or anti-gH antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-complex I antibodies and/or anti-gH antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

The antibodies in the compositions of the invention may be used alone or in combination with other active agents in therapy. For example, an antibody of the invention can be co-administered with at least one additional therapeutic agent. In particular embodiments, the additional therapeutic agent is ganciclovir, valganciclovir, foscarnitin and/or silurovir. In other embodiments, the additional therapeutic agent is an additional therapeutically isolated antibody.

The above-mentioned combination therapies encompass the administration of the combination (wherein the two or more therapeutic agents are comprised in the same or separate formulations) and the separate administration, in which case the administration of the antibody composition of the invention may be performed before, simultaneously with and/or after the administration of the further therapeutic agent and/or adjuvant.

The compositions of the invention (and any additional therapeutic agent) may be administered by any suitable method, including parenteral, intrapulmonary and intranasal, and intralesional administration if local treatment is desired. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, e.g., intravenous or subcutaneous injection, depending in part on whether administration is transient or chronic. Various dosing regimens are contemplated herein, including, but not limited to, single or multiple administrations over multiple time points, bolus administrations, and pulsed infusions.

The compositions of the present invention will be formulated, administered and administered in a manner consistent with good medical practice. Factors to be considered herein include the particular condition to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the condition, the site of active agent delivery, the method of administration, the timing of administration, and other factors known to medical practitioners. The composition need not be, but may optionally be formulated with one or more active agents currently used for the prevention or treatment of the condition in question. The effective amount of such other active agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as described herein, or at about 1-99% of the dosages described herein, or at any dosage and any route determined to be appropriate by experience/clinic.

For disease prevention or treatment, the appropriate dosage of the antibody contained in the compositions of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prevention or treatment, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. Each of the antibodies included in the compositions described herein is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for each antibody, about 1 μ g/kg-15mg/kg (e.g., 0.1mg/kg-10 mg/kg) may be an initial candidate dose for administration to a patient, whether, for example, by one or more divided administrations, or by continuous infusion. A typical daily dose may be from 1. mu.g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, treatment generally continues until the desired suppression of disease symptoms occurs. For each antibody, an exemplary dose will be about 0.05mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg, or 10mg/kg (or any combination thereof) may be administered to a patient. Such doses may be administered intermittently, such as once per week or once every three weeks (e.g., such that the patient receives about two to about twenty, such as about six doses of the antibody). An initial higher loading dose may be administered followed by one or more lower doses. The progress of this therapy can be readily monitored by routine techniques and assay tests.

It will be appreciated that any of the formulations or methods of treatment described above may be performed using immunoconjugates of the antibodies described herein instead of or in addition to anti-complex I antibodies and/or anti-gH antibodies.

I. Article of manufacture

In another aspect of the invention, there is provided an article of manufacture containing materials for the treatment, prevention and/or diagnosis of the disorders described above. An article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container contains a composition, alone or in combination with another composition effective for treating, preventing and/or diagnosing the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is for use in treating the selected condition. In addition, an article of manufacture can comprise (a) a first container having a composition therein, wherein the composition comprises an antibody of the invention; and (b) a second container containing a composition, wherein the composition comprises an additional cytotoxic agent or an additional therapeutic agent. In this embodiment of the invention, the article of manufacture can further comprise package inserts indicating that the composition can be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

It will be appreciated that any of the above-described articles of manufacture may include an immunoconjugate of an antibody described herein in place of or in addition to the anti-complex I antibody and/or the anti-gH antibody.

Example III

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be implemented in view of the general description provided above.

Materials and methods

The virus grows. VR1814 (Ravello Lab, Fondazione IRCCS Policilingo SanMatteo, Pavia Italy), expanded in 7-14 passages of human fetal pulmonary fibroblasts (MRC 5) (American type culture center, ATCC; Manassas, VA) as directed, or P0 Human Umbilical Vein Endothelial Cells (HUVEC) at 4-6 passages (Lonza; Basel, Switzerland) as directed in DMEM, the supernatant was concentrated, resuspended in complete medium and frozen. Complete medium consisted of DMEM supplemented with 10% fetal bovine serum, penicillin/streptomycin, L-glutamine (all from Invitrogen; Carlsbad, Calif.) and 10mM HEPES (Cellgro; Manassas, Va.). Assay assays were performed in MRC5 and HUVEC cells in 96-well plates and also in human retinal pigment epithelial cells (ARPE-19) (as cultured with ATCC, as directed), macrophages derived from monocytes (MDM), and cytotrophoblasts, which are placental epithelial cells. MDM was isolated from whole blood as directed using a RosetteSep Human monoclonal expression Cocktail (Stemcell Technologies, Vancover, BC, Canada). Monocytes were then stimulated with 0.1 μ g/ml Lipopolysaccharide (LPS) (Invivogen) and incubated overnight in DMEM. Platelets and unbound cells were washed out with PBS prior to infection. Cytotrophoblasts were isolated from 19-week placenta (Pereira Lab, UCSF, using protocols from Librach et al, 1991, JCV113: 437-449) and plated in 96-well tissue culture plates. The cytotrophoblast preparations were assayed for cytotrophoblast-labeled cytokeratin 7 (CK 7) (Dako) and found to be more than 90% positive at the onset of infection.

The following HCMV strains were obtained from Dr.Jay Nelson (University of Oregon Health and Science University (OHSU); Portland, OR): adinis, Brown, Cano, Davis, destin, Grunden, Harris, Keone, Lysistata, Newrock, Phoebe, Powers, Salvo, Schmoe, Simpson, and Watkins. The following HCMV strains were obtained from Dr.Sunwen Chou (OHSU): c079, C323, C327, C336, C352, C353 and C359. Thawed virus was added to MRC5 fibroblasts and allowed to grow until 100% CPE was visible (about 10-12 days post infection). After three days, cells were scraped and supernatants were harvested and concentrated using ultracentrifugation. Dilutions of each strain were used to infect fresh fibroblasts in 96-well plates. The virus was allowed to infect for 18 hours, after which the cells were fixed with 100% ethanol. Staining was performed with Mab810, an anti-IE antibody (Millipore; Billerica, Mass.) for immunofluorescence analysis. Titers were calculated and used to determine the amount of virus required for multiplicity of infection (MOI) 1 for use in the neutralization assay.

Neutralization assay test. The neutralization assay was performed essentially as described in Abai et al, 2007, j.immunol methods, 322:82-93, except that the assay was performed in dmem (gibco) and detected by immunofluorescence as described above. Briefly, antibodies were serially diluted and mixed with virus, wherein the virus was diluted in complete medium such that the final virion concentration resulted in about 1 infectious virus per cell (MOI = 1) when mixed with medium or with non-inhibitory antibodies. The antibody and virus were mixed and incubated at 37 degrees for one hour before adding to a confluent monolayer of MRC5, ARPE-19, HUVEC or Monocyte Derived Macrophages (MDM). The virus was allowed to infect for 18 hours, after which time the cells were fixed with 100% ethanol. Cells were blocked in PBS, 2% BSA and subsequently stained with anti-HCMV IE antibody, Mab810 (Millipore) or rabbit anti-HCMV IE (Johnson Lab, Oregon Health Sciences University). Cells were washed with PBS and incubated with appropriate AlexaFluor488 and Hoechst stain (Invitrogen). Data from duplicate wells containing a given antibody concentration were averaged and compared to infection in the absence of antibody (this was set at 100%). Use ofMicro^TMAnd(Molecular Devices) cells were imaged and counted. Data were log transformed, normalized, graphed, and calculated for EC50 and EC90 using Prism (GraphPad Software, La Jolla, CA). Use ofEC50 curve fitting algorithm, EC90 values were calculated from the best fit curve. The detection range of the determination test is 100-6.5x10⁵Infectious viral particles/well. Between experiments (in particular with different virus multiplicity of infection), the validation of the assay was observed.

Amplification and clustering of clinical strains. Clinical strains of HCMV (from Oregon Health sciences university) were grown on fibroblasts, supernatants were harvested, and concentrated using ultracentrifugation. DNA was isolated from infected cells using DNeasy blood and tissue kit (Qiagen). HCMV gene primers were designed using alignment of HCMV strains in the public domain. Select conserved regions that are less than 500 bases apart. Use ofSequences from clinical strains were visualized and translated using MacVector. Alignments were performed by ClustalW and in a Jalview Alignment Editor and trees were built using nearest neighbor% identity.

Proteins are expressed by baculovirus. To detect the expressed protein, rabbit polyclonal antibodies were generated by standard procedures. Each rabbit was immunized with the peptide corresponding to the HCMV protein. The peptides are as follows:

gH_871HPHHEYLSDLYTPCSSSGRRDHSLERLTR(SEQIDNO:195),

gH_977CPHVWMPPQTTPHDWKGSHTTSGLHRPH(SEQIDNO:196),

gL_873CGLPPELKQTRVNLPAHSRYGPQAVDAR(SEQIDNO:197),

UL128988VGLDQYLESVKKHKRLDVCRAKMGYMLQ(SEQIDNO:198),

UL128989RQVVHNKLTSCNYNPLYLEADGRIR(SEQIDNO:199),

UL130892RDYSVSRQVRLTFTEANNQTYTFCTHPN(SEQIDNO:200),

UL130_892SPWFTLTANQNPSPPWSKLTYPKPHDC (SEQIDNO:201), and

UL131_993TAEKNDYYRVPHYWDACSRALPDQTRYK(SEQIDNO:202).

serum was purified by capture on a peptide-bound column and subsequent elution. For secreted proteins, baculovirus constructs were prepared for the extracellular domain of each HCMV protein. The HCMV extracellular domain was fused to a baculovirus signal sequence and a 6X-His tag (on the C-terminus), respectively. Baculovirus expression vectors were used to infect SF9 or Tini insect cells. Proteins were harvested and gel filtered and examined by acrylamide gel electrophoresis and coomassie staining. Fractions containing all five proteins (gH, gL, UL128, UL130 and UL 131) were pooled. A gB trimer construct was made by fusing gB S64-K115 to Q499-E655. When expressed by infecting insect cells, the construct results in a gB protein that is not membrane anchored, forms trimers as expected, binds neutralizing and non-neutralizing gB antibodies, and is likely to be in its prefusion form. When gH and gL were co-expressed, the resulting gH/gL protein bound HB1 and the following antibodies recognizing non-conformational and conformational gH epitopes: MSL-109, rabbit anti-gH, rabbit anti-gL (from David Johnson, OHSU, Ryckman et al, J. Virol. 82:60-70 (2008)), rabbit anti-gH _977, and rabbit anti-gL _ 873. gH. gL, UL128, UL130 and UL131 co-transfect insect cells, resulting in small amounts of heterogeneous proteins that bind to hu8G8 and non-conformational and conformational antibodies, including rabbit anti-gH, anti-gL, anti-UL 128, anti-130, anti-131 and the rabbit polyclonal antibodies described above.

And constructing a pRK-CMV vector. For surface expression of full-length viral glycoproteins, three separate human expression plasmids were constructed. Each HCMV gene was amplified from the starting point to the end point. gH. gL, gB were amplified from genomic DNA and UL128, UL130, UL131 from cDNA, first cloned using PCR Blunt II TOPO (Invitrogen). Thereafter, the genes were cloned into Genentech mammalian expression vector (pRK-tk-Neo) having a "self-cleaving 2A peptide" sequence (Szymczak et al, nat. Biotechnol.22:589-94 (2004)), and each HCMV gene was separated from the last 3' gene from the gene encoding eGFP. Three plasmids were constructed, one with gB and eGFP, one with gH, gL and eGFP, and one with UL128, UL130, UL131 and eGFP. The plasmids were transfected into Human Embryonic Kidney (HEK) -293T cells (American type culture center, ATCC; Manassas, Va.) using Lipofectamine2000 (Invitrogen; Carlsbad, Calif.).

(HIG) exhaustion. For analysis of components that neutralize HIG of virus into epithelial cells, HEK-293T cells were transfected with gB/eGFP or gH/gL/eGFP and UL128/UL130/UL131/eGFP or mock plasmids using Lipofectamine2000 (Invitrogen) and incubated for 48 hours. Cells were dissociated using accutase (sigma), pelleted and divided into 12 aliquots. Will be provided withDiluted to 20. mu.g/ml in PBS and 0.5% Bovine Serum Albumin (BSA) and reacted with 3X10⁷Transfected cells were incubated in suspension for 1 hour. Then will beSeries transfer to 3X10⁷Fresh aliquots of transfected cells. Delivery on new cells until maximum specific depletion of antibody was seen (six deliveries). ELISA assay tests were performed to detect depletion of some HCMV-specific antibodies. Maxisorp (NUNC) plates were coated with purified baculovirus-produced gB, gH/gL or gH/gL/UL128/UL130/UL131 in PBS, and/or with lysates of transfected HEK293T cells. Detection was determined with goat anti-human IgG, Fc γ (jackson laboratory, Bar Harbor, ME) conjugated to horseradish peroxidase. The lower limit of detection was 0.08. mu.g/ml. The resultant HIG was then concentrated using a 100kD molecular weight cut-off concentrator (Centricon) for use in the neutralization assay test as described above.

The affinity depletion column was generated in the following manner. Proteins (1-2 mg soluble gB or gH/gL) were dialyzed against PBS extensively and added to approximately 1ml Sterogene ALDSuperflow resin equilibrated in PBS. Sodium cyanoborohydride (0.2 ml of 1M solution) was added to react the protein withThe aldehyde containing resin was chemically coupled and the reaction was allowed to proceed overnight at 4 ℃. Each resin was loaded into a column separately and washed thoroughly with PBS to remove any unbound protein. Two milligrams in a volume of 800. mu.lLoaded onto the column and washed with PBS at a flow rate of 0.4 mL/min. Unbound protein was collected into several 0.5ml aliquots, which were separately concentrated to about 100 μ l in a rotary concentrator with a molecular weight cut-off of 5000 daltons. The samples were sterile filtered and stored at 4 ℃ until the time of assay.

FACS. The previously described pRK-CMV vectors were transfected into HEK293T cells individually, or at a 50:50 ratio (for gH/gL and UL 128-131) using Lipofectamine2000 (Invitrogen). After 48 hours, the cells were dissociated using accutase (sigma). All incubations of primary or secondary antibody (Jackson Labs) and washes were in PBS, 2% FCS, 0.2% sodium azide (FACS buffer). After staining, cells were fixed in FACS buffer in 2% paraformaldehyde. Fluorescence analysis was done using FACS Calibur4 (Beckton Dickinson) and data was processed using FlowJo Software (Tree Star Inc.).

Generation of resistant viral mutants. To generate viral mutants resistant to the antibodies described herein, HCMV strain VR1814 was grown on epithelial cells during which suboptimal concentrations of antibody MSL-109 (Aulitzky et al, J.Infect.Dis.163:1344-47 (1991), which was synthesized in Genentech, HB1, hu8G8, or a combination of HB1 and hu8G8 (in ARPE19 cells (American type culture center, ATCC; Manassas, Va.) were present. The experiment was started in 24-well plates, three wells each with antibodies to EC50, 2X EC50 or EC90 and 0.5 multiplicity of infection (MOI). Every week, half of each well volume was transferred to new cells and antibody concentrations increased 1.5-fold or remained unchanged. Typically, the mutants appear as single viral plaques by approximately 9 deliveries. These viruses were grown in increasing concentrations of antibody (to a final concentration of 10 × EC 90). Subsequently, the mutants were stored and analyzed for resistance to HB1 and hu8G8 by a neutralization assay test as described above. The entire process was initiated four times apart (three wells per antibody concentration) on ARPE-19 cells and twice apart on MRC5 cells with only HB1 and MSL109 (hu 8G8 does not neutralize the virus on MRC5 cells).

To generate additional resistant mutants, extracellular viruses were treated with N-ethyl-N nitrosourea (ENU, Sigma-Aldrich; St. Louis, MO) or ultraviolet light (254. lamda Stratalinker, Stratagene; Santa Clara CA) and allowed to infect ARPE-19 or MRC5 cells in either 24 well format (24 well/treatment) or 96 well format (72 well/treatment). After infection (at MOI1 or 2), the medium was replaced with HB1 with EC100 or hu8G8 with EC100 or a combination of two antibodies of each EC50, or complete medium of ganciclovir (GCV, Sigma-Aldrich). Weekly, supernatants were passaged to fresh cells with increasing concentrations of antibody or GCV. The grown virus was transferred to larger wells and stored after 2-3 months. The whole process was initiated in two separate runs.

And (4) sequencing the glycoprotein. DNA was isolated from control or mutant virus infected cells or supernatants (DNA Blood/Tissue Extraction Kit, Qiagen; Valencia, CA). Primers for conserved sequences of each gene were designed based on alignment of HCMV strains AD169, FIX, TB40E, Toledo and Towne sequences available in the National Center for Biotechnology Information (NCBI) database in the united states. Glycoprotein H was amplified from each clinical strain, starting from the start codon to base 2196, and just lacking the stop codon. Glycoprotein B was amplified from the start codon to base 2686, and the stop codon was deleted. UL128, UL130 and UL131 were amplified from start to stop, respectively, based on the cDNA sequences obtained from Akter et al, j.gen.virol.84:1117-22 (2003). The Polymerase Chain Reaction (PCR) products were sequenced using dye termination reaction, and the sequences were aligned and trimmed (sequencers).

And (5) reenactment of mutation. The pRK-CMV expression plasmid containing the gH/gL gene or UL128/UL130/UL131 gene as described above was modified to replace the single mutation found in each of the resistance mutants. Each gH/gL plasmid was transfected (Lipofectamine 2000, Invitrogen; Carlsbad, Calif.) into HEK293T cells (ATCC), allowed to express for 2 days, and then analyzed for the ability of surface-expressed HCMV proteins to bind to control anti-gH antibodies 10F8 or HB1 by Fluorescence Activated Cell Sorting (FACS) as described above. Each UL128/UL130/UL131 plasmid was co-transfected with the gH/gL plasmid, allowed to express for 2 days, and then the surface-expressed proteins were evaluated for their ability to bind HB1, hu8G8, and control rabbit anti-UL 131_993 antibodies. Analyses and images were generated using FlowJo (Treestar; Ashland, OR).

Analysis of viral entry. Stocks of growth-resistant and control strains (passaged in parallel on ARPE-19 cells in the absence of antibody) were grown and supernatants harvested (neat). For pp65DNA (pp 65F TCGCGCCCGAAGAGG (SEQ ID NO: 189), pp65RCGGCCGGATTGTGGATT (SEQ ID NO: 190), Taqman probe CACCGACGAGGATTCCGACAACG (SEQ ID NO: 191), quantitative PCR (qPCR) WAs performed for copy number determination, standard curves were obtained with pp65 cloned into Zero Blunt PCR Cloning (Invitrogen), ARPE-19 and MRC5 cells were allowed to infect for 18 hours based on copy number viral dilution, then fixed and visualized, infectious particle number per DNA copy WAs calculated, normalized to strains passaged without antibody and plotted using Excel version 14.1.2 (Microsoft; Redmond, WA).

Example 1 Generation of anti-Complex I and anti-gH antibodies

Production of anti-complex I murine mAb8G 8. Group 2 Balb/c mice (10 in each group) were treated with fully UV-inactivated (3000 mJ) HCMV (strain VR 1814) at 1X10⁶pfu/mouse concentrations were immunized twice weekly for a total of 7 injections of SC/IP. In the first group of animals, each mouse was primed with RIBI adjuvant, followed by injection of HCMV in PBS. In the second group of mice, animals were not primed, followed by injection with HCMV in RIBI adjuvant. Serum sample titration by ELISA and the diseases described above were performed on test blood taken from immunized miceNeutralization assay. The top 5 mice with the highest response were selected for hybridoma production. Two separate fusions were performed using lymphocytes from the popliteal and inguinal lymph nodes and the mouse myeloma line X63-ag8.653. The fused cells were seeded in 96-well tissue culture plates (58 plates) and hybridoma selection was performed using HAT medium supplement (Sigma, st. louis, Mo.) starting one day after fusion. A total of 738 IgG + hybridomas were screened on epithelial cells using a virus neutralization assay as described above. The resulting antibodies were tested for EC50 (μ g/ml) on HCMV strain VR1814 for a number of cell types and compared to MSL-109 (anti-gH antibody) and are shown in table 2. Monoclonal antibody 8G8 was the most potent neutralizing antibody identified in the screen, which was selected for humanization and further characterization.

TABLE 2

Humanization and analysis of murine 8G8 mAb. Murine hybridoma 8G8 was humanized by standard CDR grafting using a λ 3 or4 light chain (fig. 2) and a VH1, VH3 or VH7 heavy chain framework (fig. 1). For comparison, an alignment of consensus human λ germline sequences for λ 3 and λ 4 is shown in fig. 2. A neutralization assay was performed comparing 8G8 human/murine chimeric antibody (QE 7/C2) with 8G8 λ 3 or λ 4 light chain in combination with 8G8VH1, VH3 or VH7 humanized heavy chains. The λ 4 variant, but not the λ 3 variant, was found to neutralize HCMV (fig. 3).

The HVR-L2 of λ 4 was mutated as shown in figure 4, introducing substitutions at amino acids 50C, 50D, 56, and an amino acid substitution at amino acid 57 (the first amino acid of FR 3), according to Kabat numbering, to provide stability of the antibody. The various mutated light chains were then combined with 8G8 human VH1 chain and the resulting antibodies were tested in a neutralization assay as described above. Antibodies with a single amino acid substitution all showed good neutralizing activity (i.e. a1, E1, T1, a2, E2 and T2) (fig. 5). Likewise, all antibodies containing two amino acid substitutions showed good neutralizing activity (i.e., SGSG and TGDA). Single mutant SG was included as a comparative control, which also showed good neutralizing activity. (FIG. 6).

The humanized 8G8 λ 4 antibody sequence is shown in figure 7 (hu8g8. λ 4 FW). Figure 8 shows the sequence of a humanized 8G8VH1 sequence (hu8g8. vh1) and figure 9 shows the sequence of a humanized 8G8VH3 sequence (hu8g8. vh3). Figure 10 shows a humanized 8G8 λ 4 antibody sequence in which the first two amino acids (QP) have been modified such that the polypeptide starts with serine (Q is deleted, L is mutated to S) and amino acid 36 retains the murine amino acid (Y). The polypeptide sequence of this antibody is shown as a λ 48G8 graft. Representative nucleic acid sequences encoding the polypeptides are shown below the polypeptide sequence.

Affinity maturation of anti-gH antibodies. Monoclonal antibody MSL-109 was synthesized using the antibody sequences for the variable heavy and variable light chain sequences of MSL-109 disclosed in PCT publication WO94/16730 (published at 8/4 of 1994, incorporated herein by reference in its entirety). The amino acid sequences of the MSL-109VH and VL chains are shown in FIG. 11 (VL, SEQ ID NO: 90; VH, SEQ ID NO: 92). The MSL-109 antibody is based on an IgG1 framework containing heavy chain VH3 and light chain V κ 2. Recombinant DNA encoding the antibody was cloned into CHO cells.

Antibody MSL-109 was affinity matured by randomization of Complementarity Determining Regions (CDRs) followed by phage display selection of binders with progressively limiting concentrations of biotinylated gH/gL. Each position of the CDRs was randomized by oligonucleotide-directed mutagenesis using "NNK" codons, where N is any of the four natural nucleotides and K is 50% thymine and 50% guanine. The NNK codon can encode any one of the 20 natural amino acids. Libraries of light and heavy chains were prepared separately and 3 CDRs from each chain were randomized simultaneously. This resulted in clones with 0-3 random amino acid changes in each strand, with no more than one mutation in each cDNA. The library was prepared by standard methods in a phage Fab fragment display vector. Binding clones were selected by incubating the phage display library with 1 and 0.1nM biotinylated gH/gL in successive rounds of selection, and then competed with 100nM gH/gL or MSL-109IgG to reduce binding of lower affinity clones to gH/gL. Bound clones were captured on ELISA plates coated with neutravidin (neutravidin) or streptavidin, washed, and eluted in 10mM HCl for 10 min at room temperature. Eluted phage were neutralized with 1/10 volumes of 1M Tris pH8.0 for infection into E.coli for amplification for the next round of selection. Clones from the second round of selection were sequenced to determine frequent mutations in the selected phage. Clones with favorable mutations were examined by competitive phage ELISA.

IgG and Fab fragments expressing mutant MSL-109 with single or combined mutations in heavy chain Kabat positions 53 and 55 were tested for in vitro neutralization of CMV. Amino acid substitutions at amino acid 53 (replacing D53 with S, I, N, Q, F, M, L, G, H, K, W, Y, V or a) alone or in combination with amino acid substitutions at amino acid 55 (replacing T55 with R or K) provided antibodies with improved neutralizing ability (fig. 12B and 12C). A schematic of some of these variations is shown in fig. 12A. In addition, amino acid N52 in MSL-109 can be replaced by S. This substitution does not affect potency but allows S to be glycosylated at position 53 without position 52. With various amino acid substitutions at amino acid 53 and/or amino acid position 55, there are 89 possible combinations for the heavy chain variable sequence (SEQ ID NOs:87, 88, 89 and 96-182). SEQ ID NO 94 provides a consensus sequence. The Fab fragments of these anti-gH antibodies were measured for affinity to gH/gL dimers produced in baculovirus in a phage display ELISA assay. Specifically, phage clones displaying MSL-109 variant Fab fragments were incubated with serial dilutions of gH/gL and incubated for 1 hour at room temperature. Unbound phage were detected by incubating the mixture with the gH/gL coated ELISA plate wells for 10 min at room temperature. The plate was washed with PBS-T and phage bound to immobilized gH/gL were detected by incubation with anti-M13 HRP conjugate for 30 minutes followed by washing and development with TMB substrate. By non-linear regression, IC 50-the point at which 50% of the phage were free in this phage-gH/gL mixture was calculated. IC50s was in the range of 0.01-0.1 nM. The affinities for the selected variants are shown in table 3.

TABLE 3

Surprisingly, amino acid changes on VH2 HVRs produced antibodies with significantly higher binding and neutralizing capacity. For example, HB1 (D53N/T55R) has a 10-fold increased affinity for gH/gL over MSL-109 as shown by phage ELISA (Table 3). As shown by the neutralization assay (fig. 12B), HB1 (D53N/T55R) was about 40-fold more potent in inhibiting HCMV entry into ARPE-19 cells than the parent MSL-109 antibody when expressed as a Fab fragment in e.coli (i.e., EC50=0.15nM versus 6.2 nM). Furthermore, HB1 (D53N/T55R) was about 6-fold more potent in inhibiting HCMV entry into ARPE-19 cells when expressed as full-length IgG in CHO cells, as shown by neutralization assay tests (table 4, fig. 12C). The EC50 and EC90 (μ g/ml) of the HB1 antibody compared to the MSL-109 antibody on various cell types in a neutralization assay (one representative experiment) are shown in Table 4 below.

TABLE 4

Example 2 functional study of antibodies

To block the ability of HCMV virus to enter epithelial cells, endothelial cells, macrophages and fibroblasts, HB1 (D53N/T55R) and hu8G8 were compared to HIG in a neutralization assay. hu8G8 had an EC50 of 0.003. mu.g/ml (0.02 nM) on epithelial cells, an EC50 of 0.004. mu.g/ml (0.03 nM) on endothelial cells and 0.001. mu.g/ml (0.006 nM) on monocytes. On each of these cell types, hu8G8 was at least 8-fold stronger than HB1 for neutralizing HCMV. However, as expected, hu8G8 did not block viral entry into fibroblasts, whereas HB1 blocked viral entry and had an EC50 of 0.11 μ G/ml (0.7 nm) (see fig. 13).

HIG has been reported to prevent HCMV fetal infection and/or disease when given to pregnant women with primary HCMV infection (Nigro et al, 2005), suggesting the ability of CMV-specific antibodies to confer protection to the developing fetus. When assessed by a neutralization assay, HIG was found to neutralize virus into all cell types tested, but with far less potency than either monoclonal antibody (see figure 13). This relatively low potency is due to the polyclonal nature of HIG, where only a small fraction of the protein has anti-CMV neutralizing activity.

Example 3-HIG depletion study.

To identify the neutralizing antibody component in the hyperimmune globulin, HIG was depleted of anti-gB antibodies or anti-complex I (gH/gL/UL 128/UL130/UL 131) antibodies by six series of incubations using gB or complex I transfected HEK293T cells. According to the method described above, performing(HIG) exhaustion. Analysis of the post-uptake sera as determined by purified gB ELISA showed that reaction with gB-transfected cells was taken up by this procedure compared to 0% on control cells>95% antibody. However, as described above, only about 45% of the antibodies that reacted with complex I-transfected cells had been taken up compared to 0% on control cells, as determined by ELISA with lysates from transfected HEK293T cells.

The depleted HIG was then used in a neutralization assay to determine the effect of depletion on preventing viral entry into epithelial cells. Serial dilutions of the absorbed HIG preparation were used in the neutralization assay test as described above, compared to the simulated absorbed HIG. The results of these experiments are shown in fig. 14. Antibodies against gB appear to have no significant contribution to the neutralizing capacity of HIG on epithelial cells, whereas antibodies against complex I appear to have a significant contribution to the neutralizing activity of HIG. Removal of complex I-specific antibodies reduced the neutralizing capacity (EC 50) of HIG by about 85% when tested on epithelial cells.

Assay of depleted HIG on fibroblasts is not possible because very high concentrations are required to detect neutralization. The EC50 for HIG was about 500. mu.g/ml for this cell type. Since UL128, UL130 and UL131 proteins are not required for entry into fibroblasts, baculovirus-expressed gB or gH/gL bound to the column as described above was used to deplete antibodies specific for these proteins/complexes from HIG. With almost complete depletion of anti-gB antibodies (95% depletion of gB antibodies on the gB column versus 0% depletion of gB antibodies on the gH/gL column), no shift in neutralization was observed. However, with the depletion of most anti-gH/gL antibodies (84% depletion of gH/gL antibodies on the gH/gL column relative to 0% depletion of gH/gL antibodies on the gB column), a 65% reduction in EC50 was observed (see fig. 14).

From these data, it was concluded that: most of the neutralizing antibodies in HIG were directed to the gH/gL/UL128/UL130/UL131 complex. In particular, for epithelial cell entry, complex I neutralizing antibodies are the primary neutralizing antibodies in HIG. In addition, the gH/gL antibodies in HIG have a major role in inhibiting viral entry into fibroblasts. These experiments showed that anti-gB antibodies had little effect in HIG neutralization.

HIG uptake by using baculovirus-expressed gB, gH/gL and gH/gL/UL128/UL130/UL131, determined by ELISA: about 1% of the HIG is gB reactive, and about 0.1-0.2% is Complex I or gH/gL reactive. By knowing the concentration of complex-specific antibodies in the HIG, the neutralizing potency of these complex-specific antibodies can be calculated by correcting the neutralizing potency of the intact HIG relative to the percentage of IgG that actually caused neutralization to the relevant complex (e.g., 810 μ g/ml x0.1-0.2=0.8-1.6 on fibroblasts), as shown in table 5 below.

TABLE 5

EC90 values were adjusted relative to the concentration of gH/gL/UL128/UL130/UL131 antibody in HIG (0.1-0.2%)

As shown in table 5 above, the combination of HB1 and humanized 8G8 (VH 1 or VH 3) was able to approach the neutralizing potency of HIG on all cell types tested in the neutralization assay. Cells were infected at the following HCMV multiplicity of infection (MOI): epithelial MOI =1, endothelial MOI =1, macrophage MOI =0.5 and fibroblast MOI = 1. HB1 has a neutralization potency comparable to HIG for inhibiting infection of fibroblasts (as corrected for the amount of HIG specific for complex I), but does not provide sufficient potency for epithelial cells, endothelial cells or macrophages. Humanized 8G8 (VH 1) and (VH 3) had comparable neutralizing potency to HIG on epithelial cells, endothelial cells and macrophages (as corrected for the amount of HIG specific for complex I). However, it fails to neutralize infection of fibroblasts. Thus, for all cell types tested, the combination of antibodies can provide HCMV neutralization comparable to HIG neutralization corrected for complex I-specific antibodies.

HB1 and hu8G8 with VH1 were again tested in neutralization assay tests for their ability to neutralize HCMV on all different cell types and compared to calculated and actual HIG neutralization potency. Cells were infected with the following HCMV MOIs: epithelial MOI =1, endothelial MOI =0.25, macrophage MOI =0.25 and fibroblast MOI = 0.8. The results of this experiment are shown in table 6 below. The average EC90 from this experiment and the results shown in table 5 are shown in the shaded box of table 6 below.

TABLE 6

HIG adjusted relative to the concentration of antibody gH/gL/UL128/UL130/UL 131.

Example 4 neutralization of HCMV clinical isolates

The gH, gL, UL128, UL130 and UL131 genes were sequenced from more than 20 clinical isolates obtained from two laboratories of the Oregon Health Sciences University and compiled with additional publicly available sequences. The publicly available sequences are generated from strains originating in the United states, Europe, and Japan.

Infected cells of each strain were lysed and DNA was extracted using a DNA blood/tissue extraction kit (Qiagen; Germanown, Md.). Primers for conserved sequences were designed based on alignments of AD169, FIX, TB40E, Toledo and Towne sequences available in the National Center for Biotechnology Information (NCBI) database in the united states. Glycoprotein gH was amplified from each clinical strain, starting from the start codon to base 2196, and just lacking the stop codon. The Polymerase Chain Reaction (PCR) products were sequenced using a dye-terminated reaction at Genentech. The sequences were aligned and trimmed (sequencers). Additional gH sequences were obtained from the NCBI database by using one of the accession numbers from Chou et al, j.infect.dis.166:604-7 (1992) and subsequently obtaining the "related sequences". Collectively, glycoprotein sequences from 57 strains were clustered after removal of the signal peptide (ClustalW, European Bioinformatics Institute (EBI); Cambridge, England) and aligned into trees using average distance by percent identity (JalView; Waterhouse et al 2009).

Sequencing results indicated that there was a 1% variation in sequence between these clinical isolates (after removal of the signal peptide) for UL128, UL130 and UL 131. This finding is consistent with a study in europe that demonstrates that UL128, UL130 and UL131 are highly conserved among pregnant women with primary HCMV infection (baldant et al, arch. virol.151:1225-33 (2006)).

gH is at least 95% identical at the protein level (after removal of the signal peptide) in all strains. A phylogenetic tree with two different branches was constructed (data not shown). This tree is consistent with previous reports in which the gH protein sequence was separated into two phylogenetic groups (Chou, j. infect. dis.166:604-7 (1992)). Also consistent with the literature, neither HCMV isolate was geographically distinguishable in both arms (i.e., strains isolated in Japan could appear in both arms) (Pignatelli, J.Gen.Virol.84: 647-.

The ability of HB1 (D53N/T55R) to neutralize the infectivity of a range of different HCMV clinical isolates on fibroblasts was tested. Table 7 shows HB1 effectiveness compared to HIG. HB1 was found to neutralize each of these HCMB strains (representing the maximum gH sequence diversity) as well as or better than HIG (when corrected for the amount of HIG specific for gH/gL/UL128/UL130/UL 131-). The HCMV strains Dement, Adinis and VR1814 were used at multiple MOIs in multiple experiments and the results of the neutralization assay tests obtained are also shown in table 7 below.

TABLE 7

EC90 values were adjusted relative to the gH/gL/UL128/UL130/UL131 antibody concentration in HCMV-HIG (0.1-0.2%)

Example 5 specificity of the antibodies

HB1 and hu8G8 were evaluated for antigen specificity. Plasmids containing viral glycoproteins were constructed so that each protein was expressed at equal stoichiometry by separating the genes with "self-cleaving 2A peptide" (Szymczak et al, nat. Biotechnol.22:589-94 (2004)). The plasmid contained the full length gene of gB/eGFP, gH/gL/eGFP or UL128/UL130/UL131/eGFP (cloned from cDNA). The plasmid was transfected into Human Embryonic Kidney (HEK) 293T cells (American type culture center, ATCC; Manassas, Va.) using Lipofectamine2000 (Invitrogen; Carlsbad, Calif.) to express CMV glycoprotein on its surface. After 2 days, cells were dissociated and stained with saturated primary anti-HB 1, hu8G8, anti-gB, affinity purified rabbit anti-UL 131_933, or affinity purified rabbit anti-gH _ 977. Cells were stained with a suitable secondary antibody conjugated to allophycocyanin (APC, Jackson ImmunoResearch; West Grove, Pa.). Fluorescence of each cell was measured using FACSCalibur (BD Biosciences; San Jose, Calif.) and analyzed using FlowJo software (Tree Star; Ashland, OR). GFP positive cells (cells expressing CMV transgene) were selectively graphed to show antibody binding.

As shown in FIG. 15, HB1 reacted with cells expressing gH/gL alone or complexed with UL128/UL130/UL 131. Hu8G8 reacted only with cells expressing gH/gL/UL128/UL130/UL131 (FIG. 15), and not with cells expressing gH/gL or gH/gL/gO alone (data not shown). None of the antibodies reacted with gB expressing cells. Thus, HB1 recognizes an epitope on gH that is present in gH/gL complex and complex I. The hu8G8 antibody binds to an epitope in the five envelope proteins that form complex I, but not to gH/gL/gO or gH/gL alone.

Example 6 evaluation of synergy or antagonism

HB1 and hu8G8 were tested in combination in a virus neutralization assay to determine if there was a difference in potency when combining the two antibodies. Since these antibodies have different targets and are presumed to act independently in blocking viral entry, the effects of HB1 and hu8G8 are assumed to be additive. Thus, the Bliss independent equation (combined reaction C for two single compounds with effects a and B is C = a + B-a × B) is applied. To this end, HB1 and hu8G8 were mixed in a 1:1 ratio and tested in a dilution series on epithelial cells in a virus neutralization assay, and EC50 was calculated as described above. HB1 did not enhance or reduce the efficacy of hu8G8 on epithelial cells, and the 1:1 curve exactly overlaid the simulated Bliss independence curve, suggesting additivity rather than synergy (see fig. 16 and table 8). Likewise, since hu8G8 did not block HCMV entry into fibroblasts (data not shown), hu8G8 did not alter the potency of HB1 on fibroblasts, as expected. Thus, HB1 and hu8G8 did not show any antagonistic or synergistic effect at a 1:1 ratio.

TABLE 8

To assess whether HB1 and hu8G8 show synergy or antagonism over a broad range of ratios, a paired "check plate" dilution series across EC50 values was used to perform neutralization assay experiments. Each antibody was diluted as shown in table 9 below, while the virus concentration was constant. The percentage of infected cells on epithelial cells (normalized to no antibody control) for various antibody concentration combinations is shown in table 9 below:

TABLE 9

Shaded portion = the concentration of each antibody where there is partial neutralization.

EC50s was determined for HB1 and hu8G8 in the presence of 0.8, 0.016, 0.0032 μ G/ml of another antibody using the neutralization assay test described above. The results of these experiments are shown in tables 10 and 11 below and in fig. 17.

TABLE 10 HU8G8 potency in combination with different concentrations of HB1

Antibody and concentration	EC50 of hu8G8 (μ G/mL)
		0.08. mu.g/ml HB1	0.0037
0.016. mu.g/ml HB1	0.0047
		HB1 of 0.0032. mu.g/ml	0.0028
0 μ g/ml HB1	0.0031

TABLE 11 HB1 potency in combination with different concentrations of hu8G8

Antibody and concentration	EC50 of HB1 (μ g/mL)
		0.0032. mu.g/ml of hu8G8	0.035
0.00064. mu.g/ml of hu8G8	0.043
		0.000128. mu.g/ml of hu8G8	0.041
0. mu.g/ml of hu8G8	0.030

There was no evidence of synergy or antagonism after comparing the potency of each antibody alone with the combination of ratios. For example, after comparing the potency curve of hu8G8 with the potency curve of hu8G8 plus 0.08 μ G/ml HB1 (infection in the absence of titrated antibody is normalized to 100%), we found that the curves overlap (see fig. 17). In turn, the respective HB1 efficacy curves overlapped (normalized to 100% infection) in the presence of different concentrations of hu8G8 (see fig. 17). Thus, there was no evidence of synergy or antagonism between the antibodies over the wide ratio range.

Example 7 evaluation of the developed Virus resistance

Although human CMV has a relatively low mutation rate (compared to hepatitis virus c (hcv) or Human Immunodeficiency Virus (HIV)), the ability of the virus to escape neutralization of HB1 or hu8G8, or both, via the generation of resistance mutations was evaluated. The virus was grown in the presence of sub-optimal concentrations of HB1 (or MSL-109), or hu8G8, or a combination of HB1 and hu8G8 antibodies. The concentration of each antibody was gradually increased as the virus was passaged onto new cells weekly (with approximately 50% of the volume transferred onto new cells per round). Mutant viruses were observed that were resistant to each antibody individually, but no mutants conferred resistance to the combination. All resistant viral mutants emerged from single plaques.

Mutants were present which conferred resistance to neutralization by HB1 (see fig. 18). However, these mutants were still sensitive to hu8G8, with similar EC50 as shown by the neutralization assay test (see figure 18 and tables 12 and 13).

TABLE 12 emergence of HCMV mutants resistant to HB1

	HB1EC50（μg/ml）
		Ab-free control	0.04
HB 1-mutant Virus 1	0.21
		HB 1-mutant Virus 2	Without neutralization
HB 1-mutant Virus 3	0.07
		HB 1-mutant Virus 4	200
HB 1-mutant Virus 5	Without neutralization
		HB 1-mutant Virus 6	Without neutralization

TABLE 13-HCMV HB1 resistance mutant remains sensitive to hu8G8 neutralization

Furthermore, mutants were present that conferred resistance to the neutralization of hu8G8, and these mutants were still sensitive to HB1, with similar EC50s (see fig. 19 and tables 14 and 15).

TABLE 14-emergence of HCMV mutants resistant to hu8G8

	hu8G8EC50（μg/ml）
		Ab-free control	0.002
hu8G 8-mutant Virus 1	Without neutralization
		hu8G 8-mutant Virus 2	0.25
hu8G 8-mutant Virus 3	Without neutralization

TABLE 15-HCMV hu8G8 resistant mutants remain sensitive to HB1 neutralization

	HB1EC50（μg/ml）
		Ab-free control	0.22
hu8G 8-mutant Virus 1	0.18
		hu8G 8-mutant Virus 2	0.07
hu8G 8-mutant Virus 3	0.06

To understand the molecular nature of resistance to HB1 and hu8G8 antibodies, gB, gH, gL, UL128, UL130 and UL131 from each resistant strain were sequenced. All HB1 resistant strains had a single non-conservative amino acid mutation in gH compared to VR1814 and D1 strains (parallel passage of VR1814 on epithelial cells without antibody pressure) and no other mutations were found in other glycoproteins (table 16). 11 strains were generated that were resistant to HB1, encompassing 5 different nucleotide mutations in only 3 amino acids. None of these amino acids were found to be mutated in the sequenced or published clinical strains of sequences.

Mutations in response to hu8G8 appeared less frequently, with only three strains found to be resistant. All three strains resistant to hu8G8 had a single non-conservative amino acid mutation in UL131, and no other mutations were found in other glycoproteins, compared to VR1814 and D1 strains (see table 16). None of these strains carried these mutations when the mutated gH and UL131 sequences were compared to the sequences of 60 available clinical strains.

TABLE 16 mapping of resistance-causing mutations

Resistant strain	Mutant proteins	Residue change
			HB 1-mutant 1	gH	P171H
HB 1-mutant 2	gH	W168C
			HB 1-mutant 3	gH	P171S
HB 1-mutant 4	gH	D446N
			HB 1-mutant 5	gH	W168C
HB 1-mutant 6	gH	W168R
			hu8G 8-mutant 1	UL131	Q47K
hu8G 8-mutant 2	UL131	K51E
			hu8G 8-mutant 3	UL131	D46N

When comparing the ability of HB1 resistant strains to infect cells relative to strain D1, these resistant strains had significant entry defects (as low as 20x potency), suggesting that these strains will be attenuated for in vivo growth (see fig. 20). When comparing the ability of the hu8G8 resistant strain to infect cells relative to the D1 strain, it was found that the resistant strain can infect cells with equal efficacy; however, these strains were very slow growing, suggesting production defects (data not shown). Further analysis is needed to understand the mechanism of this attenuation.

To determine whether these mutations affect the ability of HB1 and hu8G8 to bind to gH and UL131, respectively, complex I with resistance mutations (gH/gL/UL 128/UL130/UL 131) were transiently expressed on the surface of HEK-293T cells by site-directed mutagenesis and FACS analysis of antibody binding was performed. The mutation of P171 (mutants 1 and 3: P171 to H or S) had only two to five times higher resistance to HB1, and the binding of this antibody to gH/gL was not significantly altered. However, the mutations (W168 to C or R) found in HB1 mutants 2,5 and 6 completely abolished the ability of HB1 to bind (see fig. 21). Furthermore, these viral mutants were not neutralized by HB1 (see fig. 18). Mutant 4 (D446N) displayed an intermediate phenotype; it was 500-fold more resistant to neutralization by HB1 (see fig. 18), but binding to HB1 could still be detected (see fig. 21).

To determine how mutations in UL131 affect hu8G8 binding, HEK-293T cells were transfected with wild-type or mutant complexes of gH/gL/UL128/UL130/UL131 and binding to anti-gH (HB 1 and MSL-109), anti-UL 131_993, and hu8G8 was measured by FACS analysis (see FIG. 22). All three UL131 mutations abolished the binding of hu8G8 to the gH/gL/UL128/UL130/UL131 complex.

The HB1 resistance mutation was mapped on a structural model of HCMV glycoprotein H (based on the recently resolved structure of HSV-2gH and EBV gH) (Backovic et al, PNAS, 197:22635-22640 (2010)). All mutated residues mapped to the same surface of gH (data not shown). HB1Fab was modeled on the gH structure. The footprint (footprint) of the HB1Fab encompasses all mutations, suggesting that HB1 binds to the epitope defined by these mutations. Similarly, the hu8G8 resistance mutations were close together (four residues apart) and although the structure of UL131 was unknown, these mutations all mapped to the putative alpha-helical domain and predicted to lie on the same face of the helix. Thus, together with the binding analysis, the resistance mutations elucidate the epitopes of HB1 and hu8G8 on the gH/gL/UL128/UL130/UL131 complex.

Example 8 affinity assay

The affinities of HB1 and hu8G8 were determined by biacore and Scatchard analysis, respectively, as described below.

The affinity of HB1 for soluble baculovirus expressed gH/gL was determined by biacore analysis and was found to be 1 nM. Specifically, the ability of HB1 to bind the baculovirus expressed secreted gH/gL was assessed by Surface Plasmon Resonance (SPR) measurements (Karlsson et al 1991) using a BIAcore3000 instrument (GE Healthcare; Piscataway, NJ). SPR-based biosensors report changes in refractive index near the surface. SPR can be used to monitor the non-covalent interactions of binding partners ("analytes") injected onto a surface when protein targets ("ligands") are covalently immobilized on the sensor chip surface; real-time measurement of analyte binding can be used to determine the kinetics and affinity of the interaction.

In the case of a 1:1 interaction (where analyte B binds to immobilized ligand A), the equilibrium can be described using equation 1:

in formula 1, k_onIs the binding rate constant, k_offIs the dissociation rate constant and the equilibrium dissociation constant K_DFrom K_D=k_off/k_onAnd (4) determining. The rate of complex formation for 1:1 binding was determined using equation 2:

when expressed in terms of SPR signal (R), equation 2 can be written as equation 3:

in equation 3, C is the concentration of the free analyte, and R_maxIs the maximum analyte binding capacity of the surface. Similarly, for analyte B, which is capable of dimerizing in solution to form 2 binding sites, the equilibrium can be described by equation 4.

By measuring the concentration dependence of the on-rate, and the off-rate in the absence of free analyte, kinetic constants can be determined and used to calculate KD.

SPR measurements were performed using an anti-Fc capture method to non-covalently immobilize HB1, followed by injection of various concentrations of gH/gL for determination of binding kinetics. CM5 biosensor chips (BR 100014, research grade CM 5; BIAcore, Inc.) were docked (dock), primed with running buffer (10 mM HEPES (pH 7.4), 150mM NaCl and 0.01% polysorbate 20), and normalized with 70% glycerol according to the instructions provided by the manufacturer. As briefly outlined below, a mouse monoclonal anti-Human Fc Antibody (Human Antibody Capture Kit, BR-1008-39, BIAcore, Inc.) was immobilized on all four flow cells of a CM5 chip. Flow cells were activated with N-ethyl N' (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (NHS) (amine coupling kit, BR-1000-50; BIAcore, Inc.) using a7 minute activation time according to the protocol described by the manufacturer. Subsequently, the activated matrix was reacted with the capture antibody by amine coupling by injecting 60. mu.L of 25. mu.g/mL antibody diluted in 10mM sodium acetate, pH5.0 at a flow rate of 10. mu.L/min. At the end of the coupling injection, any remaining unreacted NHS groups were inactivated by injecting 35 μ L of 1M ethanolamine-HCl at a flow rate of 5 μ L/min. The amount of capture antibody covalently immobilized in this manner was estimated from the SPR signal before and after the coupling procedure, which gave a range of 8000- > 9500RU on 4 flow cells.

R of less than about 100 (any SPR or Response Unit (RU))_maxValues are generally accepted as providing good signal-to-noise ratios, without limiting the range of kinetic constants that can be determined. Preliminary experiments indicate that injection of 60 μ L of 0.13 μ g/mL HB1 at a flow rate of 30 μ L/min resulted in capture of sufficient HB1 such that about 50RU signal was observed after saturation with gH/gL. Thus, this HB1 concentration and injection protocol were used in the determination of kinetic constants.

Binding measurements were performed by capturing HB1 on flow cell 2 as described above, with flow cell 1 serving as a reference. Solutions of gH/gL were prepared in running buffer at 2-fold increasing concentrations from 0.39nM to 100 nM. 60 μ L of these solutions were injected on the sensor chip surface at a flow rate of 30 μ L/min, and sensorgrams were collected. The sensor chip was maintained at 25 ℃ and dissociation was monitored for 10 minutes after the end of injection. By injection 30 μ L of 3M MgCl₂The sensor chip surface is regenerated between bonding cycles. This injection caused the dissociation of any remaining HB1: gH/gL complex from the capture antibody. HB1 was then captured on flow cell 2 as above for the next binding cycle. A "blank" sensorgram was similarly collected for the injection of running buffer on the sensor chip.

The observed sensorgrams were prepared for kinetic analysis by first subtracting the signals measured for the reference cell. The signal resulting from the regenerated portion of the curve is removed. Sensorgrams were then zeroed by subtracting the mean RU value of the pre-analyte injection baseline. Finally, the sensorgram measured with injection of running buffer only was subtracted from the curve obtained with injection of the solution containing gH/gL. Data were analyzed according to the 1:1Langmuir binding model or the bivalent analyte model using software supplied by the manufacturer.

The total SPR signal increased with gH/gL concentration, indicating the ability of Fc-captured HB1 for antigen binding. Data analysis according to the 1:1Langmuir binding model indicated an apparent equilibrium dissociation constant (K) of 0.15nM_D) Wherein kinetic constants are shown in table 17; however, the calculated curve does not match well with the observed sensorgram, with a relatively large χ of 2.1²The value is obtained. Sensorgram observed by bivalent analyte K_DModel, better described (χ)²= 0.4), yielding an apparent K of 1.0nM_DAnd kinetic constants shown in table 18.

TABLE 17 kinetic constants calculated using the 1:1Langmuir binding model

Kon(M-1s-1)	Koff(s-1)	Rmax(RU)	KD(nM)	x2
					6.8×105	1.02×10-4	42	0.15	2.1

TABLE 18 use of divalent analyte K_DKinetic constants of model calculation

As biacore cannot be used to determine the affinity of hu8G8, Scatchard analysis was used as an alternative method. In this method, iodinated antibody is mixed with a dilution series of unlabeled antibody and competition is determined. The results were plotted to determine the affinity of the antibody using the Munson and Rodbard fitting algorithm (FIG. 23). The mean Kd was 1.27nM for HB1 and 2.03nM for hu8G8 (Table 17). Affinity measurements were also performed on the gH/gL/UL128/UL130/UL131 complex expressed on the adenovirus cell surface by Scatchard analysis for HB1 and hu8G8, and were found to be 1.27nM and 2.03nM, respectively.

Specifically, HB1 and hu8G8 were iodinated using the Iodogen method (Thermo-Fisher Scientific; Waltham, MA). Using NAP-5 column, filtering through gel, and separating¹²⁵Radiolabeled antibodies were purified in I-Na. The purified hu8G8 antibody had a specific activity of 12.30. mu. Ci/. mu.g, and the purified HB1 antibody had a specific activity of 14.66. mu. Ci/. mu.g. A50. mu.l competition reaction mixture containing a fixed concentration of iodinated antibody and a decreasing concentration of unlabeled antibody was added to a 96-well plate. Using SigmaSolution (Sigma-Aldrich; St. Louis, MO) the ARPE-19 cells transiently transfected with adenovirus expressing the protein complex gH/gL/128/130/131 were detached from the flask, fixed with paraformaldehyde, and washed with binding buffer (DMEM with 2% FBS, 50mM HEPES, pH7.2 and 0.1% sodium azide). Washed cells were added to a 96-well plate containing triplicate 50 μ L of competition reaction mixture at a density of 25,000 cells in 0.2mL of binding buffer. The final concentration of iodinated antibody was 100pM in each cell competition reaction, whereas the final concentration of unlabeled antibody was varied in the cell competition reactions, starting at 500nM, and subsequently decreased by 1: 2-fold dilution, for a total of 10 concentrations, and included zero addition of buffer-only sample. The cell competition reaction was incubated at room temperature for 2 hours, then transferred to a Millipore Multiscreen filter plate and washed four times with binding buffer to separate free from bound iodinated antibody. Filters were counted on a Wallac Wizard1470 gamma counter (PerkinElmer Life and Analytical Sciences; Wellesley, Mass.). The binding data was evaluated using New Ligand software (Genentech) using the fitting algorithm of Munson and Rodbard (anal. biochem., 7:22-39 (1980)) to determine the binding affinity of the antibody.

Watch 19

^a K_D= equilibrium dissociation constant; SD = standard deviation

Example 9 analysis of binding of hu8G8 to Complex I

To further characterize the binding of hu8G8 to complex I, an ELISA assay was performed to test whether hu8G8 can bind to the UL131 portion containing the resistance mutation as identified in example 7. Specifically, DNA encoding UL131 was amplified, from the codon for serine at position 41 to the codon for serine at position 68 (SRALPDQTRY KYVEQLVDLT LNYHYDAS (SEQ ID NO: 194), and cloned into a Restriction-Independent cloning (RIC) vector with an N-terminal His6, GST, and TEV cleavage site (DNA 654570). this part of UL131 forms a putative alpha-helix in the secondary structure of the protein.UL 131 with the mutation Q47K was also cloned, which mutation Q47K abolished the binding of hu8G8 to UL131 in Complex I (gH/gL/UL 128/UL130/UL 131). The sequence-verified construct was grown in E.coli strain Rosetta2 (DE 3). When starter cultures were grown in LB medium with 50. mu.g/ml carbenicillin at 30 ℃ protein expression in 1-L culture was induced overnight at 16 ℃ with 0.3mM IPTG. IPTG, immediately lysed in 100mM Tris pH8.0, 500mM NaCl, 5% glycerol (buffer A) containing EDTA-free protease inhibitor tablets (Roche) by sonication and cell disruptor. The lysed cells were centrifuged at 10000rpm for 40 minutes and the clarified lysate was loaded onto a gravity flow Ni-chelating affinity column (Qiagen). The column was washed with 10 column volumes of buffer a and 10 column volumes of buffer a with 50mM imidazole. Proteins were eluted with 100mM Tris pH8.0, 500mM NaCl, 5% glycerol, 500mM imidazole and immediately dialyzed into 50mM Tris pH8.0,200mM NaCl and 5% glycerol. The protein was further purified on a size exclusion column (S20010/30, GE) in 25mM Tris pH8.0,200mM NaCl and 5% glycerol.

To determine the binding of hu8G8 to the UL131 protein fragment, Maxsorb ELISA plates were coated overnight at 1 μ G, 200ng, or 40ng protein/well in carbonate coating buffer at 4 ℃. After 3 washes with wash buffer (PBS with 0.05% Tween20[ Sigma Chemical ]), the wells were blocked with assay diluent (wash buffer with 0.5% BSA [ Invitrogen; Carlsbad, CA ]) for one hour. hu8G8 was incubated for one hour at 10. mu.g/ml or 1. mu.g/ml in the test diluent. After 3 washes, wells were incubated with 1:5000 peroxidase conjugated anti-human antibody (Jackson Immunolabs, Bar Harbor, ME) or 1:500 or 1:5000 horseradish peroxidase conjugated anti-penta-his (qiagen) for 1 hour. The results of the experiment are shown in FIG. 24, including data from ELISA plates coated with 200ng of protein and incubated with 10. mu.g/ml hu8G8.

Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and example should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific documents cited herein are specifically incorporated by reference in their entirety.

Claims (23)

1. An isolated antibody that binds HCMV complex I comprising three heavy chain hypervariable regions (HVR-H1, HVR-H2, and HVR-H3) and three light chain hypervariable regions (HVR-L1, HVR-L2, and HVR-L3), wherein:

(a) HVR-H1 consists of the amino acid sequence of SEQ ID NO 6;

(b) HVR-H2 consists of the amino acid sequence of SEQ ID NO. 7;

(c) HVR-H3 consists of the amino acid sequence of SEQ ID NO. 8;

(d) HVR-L1 consists of the amino acid sequence of SEQ ID NO. 9;

(e) HVR-L2 consists of an amino acid sequence selected from SEQ ID NOs: 10-19; and

(f) HVR-L3 consists of the amino acid sequence of SEQ ID NO: 20.

2. An isolated antibody that binds HCMV complex I comprising three heavy chain hypervariable regions (HVR-H1, HVR-H2, and HVR-H3) and three light chain hypervariable regions (HVR-L1, HVR-L2, and HVR-L3), wherein:

(a) HVR-H1 consists of the amino acid sequence of SEQ ID NO 6;

(b) HVR-H2 consists of the amino acid sequence of SEQ ID NO. 7;

(c) HVR-H3 consists of the amino acid sequence of SEQ ID NO. 8;

(d) HVR-L1 consists of the amino acid sequence of SEQ ID NO. 9;

(f) HVR-L3 consists of the amino acid sequence of SEQ ID NO. 20; and

(e) the first amino acid of HVR-L2 and the light chain variable domain framework FR3 consisted of the amino acid sequence of SEQ ID NO: 21.

3. The antibody of claim 1, wherein the antibody that binds HCMV complex I comprises a light chain variable domain framework FR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO 35, SEQ ID NO 39 and SEQ ID NO 43; and a light chain variable domain framework FR2 comprising an amino acid sequence selected from SEQ ID NO:36, SEQ ID NO:40 and SEQ ID NO: 44.

4. The antibody of claim 1, wherein the antibody that binds HCMV complex I comprises a light chain variable domain framework FR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO 37 and SEQ ID NO 41; and a light chain variable domain framework FR4 comprising an amino acid sequence selected from SEQ ID NO:38 and SEQ ID NO: 42.

5. The antibody of claim 1, wherein the antibody that binds HCMV complex I comprises a VH sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 45, SEQ ID NO 46 and SEQ ID NO 47, and a VL sequence having at least 95% sequence identity to an amino acid sequence of SEQ ID NO 48 or SEQ ID NO 49.

6. The antibody of claim 5, wherein the VH sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NO 45, SEQ ID NO 46, and SEQ ID NO 47.

7. The antibody of claim 5, wherein the VL sequence consists of the amino acid sequence of SEQ ID NO 48 or SEQ ID NO 49.

8. The antibody of claim 5, wherein the antibody that binds HCMV complex I comprises a VH comprising an amino acid sequence selected from SEQ ID NO 45, SEQ ID NO 46 or SEQ ID NO 47; and VL comprising the amino acid sequence of SEQ ID NO 48 or SEQ ID NO 49.

9. The antibody of claim 8, wherein the antibody that binds HCMV complex I comprises the VH sequence of SEQ ID NO 45 or SEQ ID NO 46 and the VL sequence of SEQ ID NO 49.

10. The antibody of any one of claims 1-9, wherein the antibody that binds HCMV complex I neutralizes 50% HCMV at an antibody concentration of 0.05 μ g/ml to 0.0007 μ g/ml or less.

11. The antibody of any one of claims 1-10, wherein the antibody is a monoclonal antibody.

12. The antibody of any one of claims 1-11, which is a human, humanized or chimeric antibody.

13. The antibody of any one of claims 1-12, wherein the antibody is an antibody fragment.

14. The antibody of any one of claims 1-10 and 11-12, wherein the antibody that binds HCMV complex I is a full-length IgG1 antibody.

15. A composition comprising the antibody of any one of claims 1-14.

16. The composition of claim 15, further comprising an additional therapeutic agent.

17. The composition of claim 15 or 16, further comprising a pharmaceutically acceptable carrier.

18. An isolated nucleic acid encoding the antibody of any one of claims 1-14.

19. A host cell comprising the nucleic acid of claim 18.

20. A method of producing an antibody comprising culturing the host cell of claim 19 such that the antibody is produced.

21. Use of an antibody or combination of antibodies according to any one of claims 1 to 14 in the manufacture of a medicament.

22. The use of claim 21, wherein the medicament is for inhibiting, preventing or treating HCMV infection.

23. The use of claim 22, wherein the medicament is for inhibiting, preventing or treating a congenital HCMV infection or a HCMV infection in a transplant recipient.